Methods and formulations for treating sialic acid deficiencies

ABSTRACT

The present invention relates to compositions and methods for treating sialic acid deficiencies comprising extended release formulations.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 13/810,068, filed on Jun. 25, 2013, which is a national stageapplication of International Application No. PCT/US2011/043910, filed onJul. 13, 2011, which claims the priority benefit of U.S. ProvisionalPatent Application No. 61/363,995 filed on Jul. 13, 2010, the content ofeach of which are hereby incorporated by reference in their entirety.

TECHNICAL FIELD

The present invention relates to extended release formulations andmethods for treating sialic acid deficiencies.

BACKGROUND

Sialic acid is the only sugar that contains a net negative charge and istypically found on terminating branches of N-glycans, 0-glycans, andglycosphingolipids (gangliosides) (and occasionally capping side chainsof GPI anchors). The sialic acid modification of cell surface moleculesis crucial for many biological phenomena including protein structure andstability, regulation of cell adhesion, and signal transduction. Sialicacid deficiency disorders such as Hereditary Inclusion Body Myopathy(HIBM or HIBM type 2), Nonaka myopathy, and Distal Myopathy with RimmedVacuoles (DMRV) are clinical diseases resulting from a reduction insialic acid production.

HIBM is a rare autosomal recessive neuromuscular disorder caused by aspecific biosynthetic defect in the sialic acid synthesis pathway.Eisenberg et al., Nat. Genet. 29:83-87 (2001). The disease usuallymanifests between the ages of 20 to 40 with foot drop and slowlyprogressive muscle weakness and atrophy. Patients may sufferdifficulties walking with foot drop, gripping and using their hands, andnormal body functions like swallowing. Histologically, it is associatedwith muscle fiber degeneration and formation of vacuoles containing15-18 nm tubulofilaments that immunoreact like β-amyloid, ubiquitin,prion protein and other amyloid-related proteins. Askanas et al., CurrOpin Rheumatol. 10:530-542 (1998). Both the progressive weakness andhistological changes initially spare the quadriceps and certain othermuscles of the face. However, the disease is relentlessly progressivewith patients becoming incapacitated and wheelchair-confined within twoto three decades. There are no treatments currently available.

Studies of an Iranian-Jewish genetic isolate mapped the mutationassociated with HIBM to chromosome 9p12-13. Argov et al., Neurology60:1519-1523 (2003). The causative mutations were identified for HIBM inthe gene GNE, which encodes the bifunctional enzymeUDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase(GNE/MNK). Eisenberg et al., Nat. Genet. 29:83-87 (2001). DMRV is aJapanese variant, allelic to HIBM. Nishino et al., Neurology59:1689-1693 (2002).

The biosynthesis steps and feedback regulation of GNE/MNK is depicted inFIG. 1. The production of sialic acid on glycoconjugates requires theconversion of N-acetylglucosamine (conjugated to its carrier nucleotidesugar UDP) to sialic acid. The sialic acid subsequently enters thenucleus where it is conjugated with its nucleotide sugar carrier CMP tomake CMP-sialic acid, which is used as a donor sugar for glycosylationreactions in the cell. CMP-sialic acid is a known regulator of GNE/MNKactivity. Jay et al., Gene Reg. & Sys. Biol. 3:181-190 (2009). Patientswith HIBM have a deficiency in the production of sialic acid via therate controlling enzyme GNE/MNK, which conducts the first two steps ofthis sequence: 1) epimerization of the glucosamine moiety to mannosaminewith release of UDP, and 2) phosphorylation of the N-acetylmannosamine.The mutations causing HIBM occur in the regions encoding either theepimerase domain (GNE) or the kinase domain (MNK). Nearly twenty GNEmutations have been reported in HIBM patients from different ethnicbackgrounds with founder effects among the Iranian Jews and Japanese.Broccolini et al., Hum. Mutat. 23:632 (2004). Most are missensemutations and result in decreased enzyme GNE activity andunderproduction of sialic acid. Sparks et al., Glycobiology15(11):1102-10 (2005); Penner et al., Biochemistry 45:2968-2977 (2006).

Knock-out of the GNE/MNK gene in mice is lethal as no sialic acid isincompatible with life, but knock-in introduction of human mutant formsof GNE/MNK have allowed the production of mouse models with humandisease features. In the DMRV-HIBM mouse model in which Gne-deficientmice transgenically express the human GNE gene with D176V mutation(Gne^(−/−) hGNED17 6V-Tg), these mice show hyposialylation in variousorgans in addition to the characteristic features of muscle atrophy,weakness and degeneration, and amyloid deposition. In these mice,hyposialylation is documented from birth, yet the mice only developmuscle symptoms several weeks later, including decreased twitch forceproduction in isolated muscles starting at 10 weeks of age andimpairment of motor performance from 20 weeks of age onward. Muscleatrophy and weakness were, however, reduced or prevented after treatmentwith administration of a sialic acid precursor N-acetylmannosamine(ManNAc), sialic acid, or sialyl-lactose, in water. Malicdan et al.,Nat. Medicine 15(6):690-695 (2009). All three sialic acid metabolitetested showed similar treatment effects. In another mouse model of HIBMin which knockin mice harbor the M712T Gne/Mnk mutation, mice homozygousfor the M712T Gne mutation died within 72 hours after birth, but lackeda muscle phenotype. Galeano et al., J. Clin. Investigation 117(6)1585-1594 (2007). Homozygous mice, however, did have severe glomerularhematuria and podocytopathy, including effacement of the podocyte footprocesses and segmental splitting of the glomerular basement membrane(GBM). Administration of ManNAc in water to mutant mice improvedsurvival, improved renal histology including less flattened and fusedpodocyte foot processes, increased sialylation of renal podocalyxin, andincreased sialylation of brain PSA-NCAM. Galeano et al., J. Clin.Investigation 117(6) 1585-1594 (2007).

Theoretically, the replacement of any metabolite after the genetic blockin the pathway could alleviate symptoms of a sialic acid deficiency ifthe production of sialic acid is the key reason the mutation causes thedisease. Jay et al., Gene Reg. and Sys. Biology 3:181-190 (2009). Thechallenge in administering a compound in the sialic acid biosyntheticpathway in vivo, however, is mainly its rapid clearance and excretion inthe urine. After a single intraperitoneal injection ofN-acetylneuraminic acid (NeuAc), the sialic acid concentration in theserum was considerably increased within minutes, but 90% of the sialicacid was found in the urine within 5-30 min, and almost all of it wasexcreted within 4 hours. After a single dose of NeuAc by theintragastric route, the sialic acid concentration in the serum was halfthat achieved by the intraperitoneal route, but the excretion rate wasslower, as 70% of the sialic acid was found in the urine within 30-60min. A similar pattern of rapid excretion was observed after a singledose of the physiological sialic acid precursor, ManNAc. Malicdan etal., Nat. Medicine 15(6):690-695 (2009).

Treatment experiments by Galeano et al. and Malicdan et al. describedabove utilized exposure to the drug via water intake which provides alonger term exposure to drug than might be achieved by bolus or episodictreatment. Continuous treatment, such as continuous water-basedexposure, is not reasonable or preferred in human treatment due in partto the logistics of performing it and the resulting difficulty withmedication compliance.

Further, the treatment of sialic acid deficiencies is complicated by thefact that each individual may carry a different GNE mutation, which mayaffect either the epimerase domain (GNE) and/or kinase domain (MNK) ofthe enzyme and to varying degrees. The residual catalytic activity ofthe MNK might perform this function, but not in all patients. If ManNAcis administered to an individual who lacks most or all MNK activity,treatment will be dependent on unknown kinases or N-acetylglucosaminekinase to transform ManNAc to ManNAc-6-phosphate. The degree andquantity of these other kinase enzymes and their efficiency intransforming ManNAc to the phosphorylated form is unknown and willlikely vary between people. This process may be slow or unpredictableand could delay or limit the onset of sialic acid production in somepatients relative to giving sialic acid directly. ManNAc may have anadvantage if its absorption into cells is improved due to its lack ofcharge. Its distribution to some tissues or in some people may be betterthan charged sialic acid if the variable kinase and metabolic steps aswell as the rapid clearance are not limiting. ManNAc is a direct productof GNE/MNK and so may also act as a typical product inhibitor of theresidual enzyme activity causing a decrease in endogenous production.This phenomenon may explain the very flat therapeutic effect curveshowing that 20 mg/kg, 200 mg/kg and 2,000 mg/kg had very similar levelsof efficacy which was still not completely effective in preventingdisease in mouse DMRV model experiments. Malicdan et al., Nat. Medicine15(6):690-695 (2009).

Sialic acid as treatment avoids the need for the random kinasephosphorylation of ManNAc and is more immediately available. Since itavoids the uncertainty of the kinase step, sialic acid may provide abetter replacement efficacy if the genetics and phenotype of the patientmake phosphorylation less efficient in a given individual and tominimize tissue-dependent phosphorylation variations. Sialic acid'saction will likely be more immediate as it is near the end of the sialicacid biosynthetic pathway; however, sialic acid is also quickly clearedprimarily by the kidneys. Therefore, sialic acid may have rapid-on andrapid-off effects. In addition, giving too much of sialic acid couldresult in a surge in CMP-sialic acid which could act as a potentialfeedback inhibitor of the GNE/MNK enzyme, which could have negativeconsequences on overall biosynthesis of sialic acid production andworsen symptoms of the sialic acid deficiency. Jay et al., Gene Reg. andSys. Biology 3:181-190 (2009).

Effective replacement of substrate within the sialic biosyntheticpathway may require a more steady even exposure to non-inhibiting orregulating levels of sialic acid metabolites, while at the same timemaintain an adequate pool of CMP-sialic acid for glycosylationreactions. Several data suggest that the production of CMP-sialic acidis highly regulated and that there is not a large pool of CMP sialicacid allowed to accumulate. The homology of mouse and human GNE/MNK isabout 98% which is far above the normal levels of homology, suggestingvery tight control of this enzyme's function. Seppala et al., Am. J.Hum. Genet. 64: 1563-1569 (1999). The allosteric regulation byCMP-sialic acid further suggests that the system is finely tuned toassure steady production of sialic acid and not excess production.Mutations in GNE/MNK that interfere with this regulation cause a diseasecalled sialuria due to excess sialic acid production. Since sialic acidis prepared for attachment to biologicals as a high energy nucleotidecarrier CMP, it is certainly expected that excess use of a high energyintermediate would not be optimal for the cell. Finally, data frompharmacokinetics and metabolism experiments suggest that very smallpercentages of administered sialic acid or ManNAc are incorporated intothe body after a dose, and the vast majority is excreted and not stored.Given the lack of a significant pool, dietary sialic acid or mannosamineis insufficient to maintain glycosylation through the day and night. Themuscle is most limited since its expression of GNE/MNK is very lowcompared to other tissues like the liver. Given the muscle phenotype ofmutations, it is reasonable to assume that biosynthesis is tightlyregulated to sialylation needs, and that no substantial pool exists invivo. In this situation, effective delivery of CMP-sialic acid to thesites of glycosylation in the cell requires a steady and continuousexposure to assure that feedback regulation is not induced by spikes inconcentration, and that glycosylation does not become deficient duringperiods of low substrate. This low period may be particularlyproblematic at night, when intake of sialic acid or metabolites is notoccurring, and ⅔rds of the growth hormone and insulin-like growth factor1 (IGF-1) is produced, which is critical for inducing muscle repair andanabolism. Frost and Lang Minerva, Endocrinol. 28:53-73 (2003);Wajnrajch J., Pediatr. Endocrinol. Metab. 18:325-338 (2005). Effectivesubstrate replacement may require then continuous and steady provisionof the metabolites to many tissues and be effective in a wide variety ofpatient types with different mutations.

Given the need for continuous exposure and the plasma half-lives ofsialic acid biosynthetic pathway components, there is a need forformulations which provide extended exposure to sialic acid, reduceimmediate surges in the metabolites of sialic acid, have general tissueavailability, and are efficacious in individuals across a broad range ofgenotypes/phenotypes.

All publications and patent applications cited in this specification areincorporated herein by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

SUMMARY OF THE INVENTION

The present invention provides extended release pharmaceuticalformulations comprising one or more compounds in the sialic acidbiosynthetic pathway or derivative thereof.

In some embodiments of any of the extended release formulations, theextended release formulation comprises two compounds in the sialic acidbiosynthetic pathway or derivative thereof. In some embodiments, the twocompounds in the extended release formulations are in a weight to weightpercent of about 50%:50%.

In some embodiments of any of the extended release formulations, the oneor more compounds comprise N-acetyl mannosamine or a derivative thereof.In some embodiments, the one of more compounds comprise N-acetylmannosamine. In some embodiments of any of the extended releaseformulations, the one of more compounds comprise sialic acid or aderivative thereof. In some embodiments, the one of more compoundscomprise sialic acid.

In some embodiments of any of the extended release formulations, the oneor more compounds comprise N-acetyl mannosamine or a derivative thereofand sialic acid or a derivative thereof. In some embodiments, theextended release formulation comprises N-acetyl mannosamine and sialicacid. In some embodiments, the N-acetyl mannosamine and sialic acid arein a weight to weight percent of about 50%:50%.

In some embodiments of any of the extended release formulations, theextended release formulation comprises one or more polymers selectedfrom the group consisting of a) at least one water-swellable, pHindependent polymer, b) at least one anionic, pH-dependent, gel-formingcopolymer, c) at least one cationic polymer, and d) at least onehydrocolloid polymer.

In some embodiments of any of the extended release formulations, theextended release formulation is capable of delivering to an individualin need thereof a therapeutically effective amount of one or morecompounds in the sialic acid pathway or derivatives thereof over aperiod of greater than about one hour. In some embodiments, the extendedrelease formulation is capable of delivering to the individual in needthereof a therapeutically effective amount of one or more compounds inthe sialic acid pathway or derivatives thereof over a period of overabout 24 hours. In some embodiments, the extended release formulation iscapable of delivering to the individual in need thereof atherapeutically effective amount of one or more compounds in the sialicacid pathway or derivatives thereof over a period of over about 12hours. In some embodiments, the extended release formulation issufficient to allow adequate replacement of sialic acid metabolitesalone or in combination during the entire night or a complete sleepcycle during the period of peak muscle repair and anabolism.

In some embodiments of any of the extended release formulations, theextended release formulation is capable of delivering to the individualin need thereof one or more compounds in the sialic acid pathway orderivatives thereof with a T_(max) of between about 2 hours and about 8hours. In some embodiments, the extended release formulation is capableof delivering to the individual in need thereof one or more compounds inthe sialic acid pathway or derivatives thereof with a T_(max) of betweenabout 3 hours and about 4 hours.

In some embodiments of any of the extended release formulations, theextended release formulation is capable of delivering to an individualin need thereof between about 0.1 g/day and about 50 g/day of one ormore compounds in the sialic acid pathway or derivatives thereof. Insome embodiments, the extended release formulation is capable ofdelivering to an individual in need thereof between about 1 g/day andabout 5 g/day.

In some embodiments of any of the extended release formulations, theextended release formulation is capable of delivering to an individualin need thereof between about 0.01 mg/kg to about 750 mg/kg of one ormore compounds in the sialic acid pathway or derivatives thereof. Insome embodiments, the extended release formulation is capable ofdelivering to an individual in need thereof between about 5 mg/kg andabout 50 mg/kg.

Provided herein are also method for treating a sialic acid deficiency inan individual in need thereof and/or preventing development of a sialicacid deficiency in an individual in need thereof comprisingadministering an effective amount of one or more compounds in the sialicacid pathway or derivatives thereof in the extended release formulationas described herein. Also provided are methods of making the extendedrelease formulations detailed herein, unit dosages of the extendedrelease formulations and kits and articles of manufacture comprising theextended release formulations. Oral dosage forms of the extended releaseformulations are also provided, such as solid or liquid dosage forms.Solid dosage forms of the extended release formulations are particularlyprovided, such as tablets or capsules.

In some embodiments of any of the methods, the sialic acid deficiency isa myopathy associated with sialic acid deficiency. In some embodiments,the myopathy associated with sialic acid deficiency is HereditaryInclusion Body Myopathy (HIBM), Nonaka myopathy, and/or Distal Myopathywith Rimmed Vacuoles (DMRV).

An extended release pharmaceutical formulation comprising one or morecompounds in the sialic acid biosynthetic pathway or derivative thereof(e.g., sialic acid and/or ManNAc or a pharmaceutically acceptable saltthereof) and one or more polymers selected from the group consisting ofa) at least one water-swellable, pH independent polymer (e.g.,hypromellose such as hypromellose type 2208 or Methocel K100M), b) atleast one anionic, pH-dependent, gel-forming copolymer (alginate salt,such as sodium alginate), c) at least one cationic polymer, and d) atleast one hydrocolloid polymer (e.g., carrageenan, such as lamdacarrageenan) is provided. In one aspect, an extended release formulationcomprises one or more compounds in the sialic acid biosynthetic pathwayor derivative thereof (e.g., sialic acid and/or ManNAc or apharmaceutically acceptable salt thereof); at least one anionic,pH-dependent, gel-forming copolymer (alginate salt, such as sodiumalginate); at least one hydrocolloid polymer (e.g., carrageenan, such aslamda carrageenan); and at least one water-swellable, pH independentpolymer (e.g., hypromellose such as hypromellose type 2208 or MethocelK100M) or cationic polymer. Such an extended release formulation mayfurther comprise microcrystalline cellulose and silicon dioxide. Such anextended release formulation may further comprise magnesium stearate.Such an extended release formulation may further comprisemicrocrystalline cellulose, silicon dioxide and magnesium stearate. Suchextended release formulations may further comprise an enteric coating.An extended release formulation may also exhibit any one or more of thefollowing characteristics: (i) be capable of delivering to an individualin need thereof a therapeutically effective amount of one or morecompounds in the sialic acid pathway or derivatives thereof over aperiod of greater than about one hour or over a period of greater thanabout 12 hours; (ii) be capable of delivering to the individual in needthereof a therapeutically effective amount of one or more compounds inthe sialic acid pathway or derivatives thereof with a T_(max) of betweenabout 2 hours and about 8 hours; (iii) be capable of delivering to anindividual in need thereof between about 0.1 g/day and about 50 g/day ofone or more compounds in the sialic acid pathway or derivatives thereof;and (iv) be capable of delivering to an individual in need thereofbetween about 0.01 mg/kg to about 750 mg/kg of one or more compounds inthe sialic acid pathway or derivatives thereof. In one aspect, theextended release formulations detailed herein are formulated for oraldelivery to an individual (e.g., is formulated as a tablet or capsule).An extended release formulation as detailed herein may be used in amethod for treating a sialic acid deficiency (e.g., myopathy associatedwith sialic acid deficiency such as any of Hereditary Inclusion BodyMyopathy (HIBM), Nonaka myopathy, and/or Distal Myopathy with RimmedVacuoles (DMRV)) in an individual in need thereof comprisingadministering the individual an effective amount of the extended releaseformulation (e.g., an extended release formulation comprising aneffective amount of a compound in the sialic acid pathway or derivativesthereof or salt of the foregoing, such as sialic acid or MaNAc). In aparticular aspect, a method of treating a sialic acid deficiency (e.g.,myopathy associated with sialic acid deficiency such as any ofHereditary Inclusion Body Myopathy (HIBM), Nonaka myopathy, and/orDistal Myopathy with Rimmed Vacuoles (DMRV)) in an individual in needthereof is provided, the method comprising administering the individualan effective amount of an extended release formulation comprising one ormore compounds in the sialic acid biosynthetic pathway or derivativethereof (e.g., sialic acid and/or ManNAc or a pharmaceuticallyacceptable salt thereof) and one or more polymers selected from thegroup consisting of a) at least one water-swellable, pH independentpolymer (e.g., hypromellose such as hypromellose type 2208 or MethocelK100M), b) at least one anionic, pH-dependent, gel-forming copolymer(alginate salt, such as sodium alginate), c) at least one cationicpolymer, and d) at least one hydrocolloid polymer (e.g., carrageenan,such as lamda carrageenan) and optionally comprising microcrystallinecellulose, silicon dioxide and magnesium stearate. In a further aspect,a method of treating a sialic acid deficiency (e.g., myopathy associatedwith sialic acid deficiency such as any of Hereditary Inclusion BodyMyopathy (HIBM), Nonaka myopathy, and/or Distal Myopathy with RimmedVacuoles (DMRV)) in an individual in need thereof is provided, themethod comprising administering the individual an effective amount of anextended release formulation comprising one or more compounds in thesialic acid biosynthetic pathway or derivative thereof (e.g., sialicacid and/or ManNAc or a pharmaceutically acceptable salt thereof); atleast one anionic, pH-dependent, gel-forming copolymer (alginate salt,such as sodium alginate); at least one hydrocolloid polymer (e.g.,carrageenan, such as lamda carrageenan); and at least onewater-swellable, pH independent polymer (e.g., hypromellose such ashypromellose type 2208 or Methocel K100M) or cationic polymer.

An extended release pharmaceutical formulation comprising one or morecompounds in the sialic acid biosynthetic pathway or derivative thereof(e.g., sialic acid and/or ManNAc or a pharmaceutically acceptable saltthereof) and one or more polymers selected from the group consisting ofa) at least one water-swellable, pH independent polymer (e.g.,hypromellose such as hypromellose type 2208 or Methocel K100M), b) atleast one anionic, pH-dependent, gel-forming copolymer (alginate salt,such as sodium alginate), c) at least one hydrogel (e.g., polyethyleneoxide such as Polyox WSR), and d) at least one hydrocolloid polymer(e.g., carrageenan, such as lamda carrageenan) is provided. In oneaspect, an extended release formulation comprises one or more compoundsin the sialic acid biosynthetic pathway or derivative thereof (e.g.,sialic acid and/or ManNAc or a pharmaceutically acceptable saltthereof); at least one anionic, pH-dependent, gel-forming copolymer(alginate salt, such as sodium alginate); at least one hydrocolloidpolymer (e.g., carrageenan, such as lamda carrageenan); and at least onewater-swellable, pH independent polymer (e.g., hypromellose such ashypromellose type 2208 or Methocel K100M) or hydrogel (e.g.,polyethylene oxide such as Polyox WSR). An extended release formulationmay further comprise microcrystalline cellulose and silicon dioxide. Anextended release formulation may further comprise magnesium stearate. Anextended release formulation may further comprise microcrystallinecellulose, silicon dioxide and magnesium stearate. Such extended releaseformulations may further comprise an enteric coating. An extended relaseformulation may also exhibit any one or more of the followingcharacteristics: (i) be capable of delivering to an individual in needthereof a therapeutically effective amount of one or more compounds inthe sialic acid pathway or derivatives thereof over a period of greaterthan about one hour or over a period of greater than about 12 hours;(ii) be capable of delivering to the individual in need thereof atherapeutically effective amount of one or more compounds in the sialicacid pathway or derivatives thereof with a T_(max) of between about 2hours and about 8 hours; (iii) capable of delivering to an individual inneed thereof between about 0.1 g/day and about 50 g/day of one or morecompounds in the sialic acid pathway or derivatives thereof; and (iv)capable of delivering to an individual in need thereof between about0.01 mg/kg to about 750 mg/kg of one or more compounds in the sialicacid pathway or derivatives thereof. In one aspect, the extended releaseformulation is formulated for oral delivery to an individual (e.g., isformulated as a tablet or capsule). An extended release formulation maybe used in a method for treating a sialic acid deficiency (e.g.,myopathy associated with sialic acid deficiency such as any ofHereditary Inclusion Body Myopathy (HIBM), Nonaka myopathy, and/orDistal Myopathy with Rimmed Vacuoles (DMRV)) in an individual in needthereof comprising administering the individual an effective amount ofthe extended release formulation (e.g., an extended release formulationcomprising an effective amount of a compound in the sialic acid pathwayor derivatives thereof or salt of the foregoing, such as sialic acid orMaNAc). In a particular aspect, a method of treating a sialic aciddeficiency (e.g., myopathy associated with sialic acid deficiency suchas any of Hereditary Inclusion Body Myopathy (HIBM), Nonaka myopathy,and/or Distal Myopathy with Rimmed Vacuoles (DMRV)) in an individual inneed thereof is provided, the method comprising administering theindividual an effective amount of an extended release formulationcomprising one or more compounds in the sialic acid biosynthetic pathwayor derivative thereof (e.g., sialic acid and/or ManNAc or apharmaceutically acceptable salt thereof) and one or more polymersselected from the group consisting of a) at least one water-swellable,pH independent polymer (e.g., hypromellose such as hypromellose type2208 or Methocel K100M), b) at least one anionic, pH-dependent,gel-forming copolymer (alginate salt, such as sodium alginate), c) atleast one hydrogel (e.g., polyethylene oxide such as Polyox WSR), and d)at least one hydrocolloid polymer (e.g., carrageenan, such as lamdacarrageenan) and optionally comprising microcrystalline cellulose,silicon dioxide and magnesium stearate. In a further aspect, a method oftreating a sialic acid deficiency (e.g., myopathy associated with sialicacid deficiency such as any of Hereditary Inclusion Body Myopathy(HIBM), Nonaka myopathy, and/or Distal Myopathy with Rimmed Vacuoles(DMRV)) in an individual in need thereof is provided, the methodcomprising administering the individual an effective amount of anextended release formulation comprising one or more compounds in thesialic acid biosynthetic pathway or derivative thereof (e.g., sialicacid and/or ManNAc or a pharmaceutically acceptable salt thereof); atleast one anionic, pH-dependent, gel-forming copolymer (alginate salt,such as sodium alginate); at least one hydrocolloid polymer (e.g.,carrageenan, such as lamda carrageenan); and at least onewater-swellable, pH independent polymer (e.g., hypromellose such ashypromellose type 2208 or Methocel K100M) or hydrogel (e.g.,polyethylene oxide such as Polyox WSR).

In another variation, a method of treating a sialic acid deficiency(e.g., myopathy associated with sialic acid deficiency such as any ofHereditary Inclusion Body Myopathy (HIBM), Nonaka myopathy, and/orDistal Myopathy with Rimmed Vacuoles (DMRV)) in an individual in needthereof is provided, the method comprising administering the individualan effective amount of an extended release formulation comprising sialicacid, or a pharmaceutically acceptable salt thereof, as the therapeuticagent. In another variation, the extended release formulation for use inthe methods detailed herein comprises MaNAc, or a pharmaceuticallyacceptable salt thereof, as the therapeutic agent. In another variation,the extended release formulation for use in the methods detailed hereincomprises sialic acid, or a pharmaceutically acceptable salt thereof andMaNAc, or a pharmaceutically acceptable salt thereof, as the therapeuticagents. In another variation, the extended release formulation for usein the methods detailed herein comprises a prodrug of one or morecompounds in the sialic acid biosynthetic pathway such as a prodrug ofsialic acid, or a pharmaceutically acceptable salt thereof, as thetherapeutic agent. In another variation, the extended releaseformulation for use in the methods detailed herein comprises sialicacid, or a pharmaceutically acceptable salt thereof, as the therapeuticagent and further comprises a hydocolloid polymer, an anionic,pH-dependent gel forming co-polymer and a water swellable, pHindependent polymer and optionally further comprises a lubricant and/oran excipient. In a particular variation, the extended releaseformulation for use in the methods detailed herein comprises sialicacid, or a pharmaceutically acceptable salt thereof, carrageenan, sodiumalginate and either hypromellose or polyethylene oxide. In a furthervariation, the extended release formulation for use in the methodsdetailed herein comprises sialic acid, or a pharmaceutically acceptablesalt thereof, carrageenan, sodium alginate, either hypromellose orpolyethylene oxide, magnesium strearate and microcrystalline colluloseand colloiddal silicon dioxide. In one aspect, the extended releaseformulation for use in the methods detailed herein is a formulation ofTable E. In yet another aspect, the extended release formulation for usein the methods detailed herein is a formulation of Table 8.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 provides a diagram of intracellular sialic acid metabolism.

FIG. 2 shows the particle size distribution for sialic acid.

FIG. 3 shows the particle size distribution plot for ProCR sialic acid250 mg final blends.

FIG. 4 shows the dissolution plot of sialic acid 250 and 325 mgsustained release (SR) tablets by direct compression

FIG. 5 shows the dissolution profile of sialic acid 325 and 500 mgsustained release (SR) uncoated tablets.

FIG. 6 shows the dissolution profile of sialic acid 325 and 500 mgsustained release (SR) coated tablets.

FIG. 7 shows the dissolution profile of ManNAc 325 mg tablets.

FIGS. 8A-8G shows the individual concentrations of sialic acid versustime in beagle dog serum following IV or oral administration. FIGS. 8Aand 8B show sialic acid concentration after administration of TA-1capsules on day 1. FIG. 8C shows sialic acid concentration afteradministration of TA-2 tablets on day 9. FIG. 8D shows sialic acidconcentration after administration of TA-3 tablet on day 13. FIG. 8Eshows sialic acid concentration after administration of TA-4 tablets onday 9. FIG. 8F shows sialic acid concentration after administration ofTA-5 tablets on day 13. FIGS. 8G and 8H show sialic acid concentrationafter intravenous administration of TA-6 on day 6.

DETAILED DESCRIPTION

The present application provides extended release pharmaceuticalformulations comprising one or more compounds in the sialic acidbiosynthetic pathway or derivative thereof and methods of treating andpreventing sialic acid deficiencies utilizing the extended releasepharmaceutical formulations. This invention concerns designing anapproach to substrate replacement that provides individuals with sialicacid deficiencies stable and steady day and nighttime replacementwithout high concentration spikes across a broad range of genotypes andin multiple tissues. This invention can optimally achieve this substratereplacement and treatment benefit through the combination of usingextended release formulations and one or more metabolites, includingcombinations of metabolites.

It is understood that the description refers to and includes effectiveamounts of an active agent, such as the compounds provided herein, whichinclude but are not limited to the compounds included under the heading“Therapeutic Agent.” Thus, it is understood that any of the extendedrelease formulations detailed herein may comprise an effective amount ofa therapeutic agent, such as an effective amount of sialic acid, or apharmaceutically acceptable salt thereof.

DEFINITIONS

The terms “oral administration” and “oral ingestion” refer to allconventional forms for the oral delivery of a pharmaceutical compositionto an individual and that result in the deposition of the pharmaceuticalformulation into the gastrointestinal tract (including the gastroportion of the gastrointestinal tract, i.e., the stomach) of thepatient. Accordingly, oral administration and oral ingestion include, byway of example, actual ingestion of a solid or liquid pharmaceuticalcomposition, oral gavage, and the like.

The terms “treating” and “treatment” as used herein refer to an approachfor obtaining beneficial or desired results including clinical results.For purposes of this invention, beneficial or desired clinical resultsinclude, but are not limited to, one or more of the following:decreasing the severity and/or frequency one or more symptoms resultingfrom the disease, diminishing the extent of the disease, stabilizing thedisease (e.g., preventing or delaying the worsening of the disease),delay or slowing the progression of the disease, ameliorating thedisease state, increasing production of sialic acid, the sialylationprecursor CMP-sialic acid (e.g., increasing intracellular production ofsialic acid) and restoring the level of sialylation in muscle and otherproteins, decreasing the dose of one or more other medications requiredto treat the disease, and/or increasing the quality of life. “Treating”a patient with a formulation described herein includes management of anindividual to inhibit or cause regression of a disease or condition.

“Prophylaxis” or “prophylactic treatment” “or preventive treatment”refers to prevention of the occurrence of symptoms and/or theirunderlying cause, for example, prevention of a disease or condition in apatient susceptible to developing a disease or condition (e.g., at ahigher risk, as a result of genetic predisposition, environmentalfactors, predisposing diseases or disorders, or the like). Prophylaxisincludes HIBM myopathy in which chronic disease changes in the musclesare irreversible and for which animal model data suggests treatmentbenefit in prophylaxis.

As used herein, “delaying” the progression of the disease means todefer, hinder, slow, retard, stabilize, and/or postpone development ofthe disease. This delay can be of varying lengths of time, depending onthe history of the disease and/or individual being treated.

As used herein, an “at risk” individual is an individual who is at riskof developing a sialic acid deficiency. An individual “at risk” may ormay not have detectable disease, and may or may not have displayeddetectable disease prior to the treatment methods described herein. “Atrisk” denotes that an individual has one or more so-called risk factors,which are measurable parameters that correlate with development of asialic acid deficiency, which are described herein. An individual havingone or more of these risk factors has a higher probability of developinga sialic acid deficiency than an individual without these riskfactor(s).

The term “effective amount” refers to the amount of a pharmaceuticalformulation including one or more compounds in the sialic acidbiosynthetic pathway that provides the one or more compounds in thesialic acid biosynthetic pathway in a sufficient amount to render adesired treatment outcome. An effective amount may be comprised withinone or more doses, i.e., a single dose or multiple doses may be requiredto achieve the desired treatment endpoint.

A “therapeutically effective amount” refers to an amount of apharmaceutical formulation including one or more compounds in the sialicacid biosynthetic pathway sufficient to produce a desired therapeuticoutcome (e.g., reduction of severity of a disease or condition). A“prophylactically effective amount” refers to an amount of apharmaceutical formulation including one or more compounds in the sialicacid biosynthetic pathway sufficient to prevent or reduce severity of afuture disease or condition when administered to an individual who issusceptible and/or who may develop a disease or condition.

The term “extended release” refers to a drug-containing formulation orfraction thereof, in which release of the drug is not immediate, i.e.,with an “extended release” formulation, administration does not resultin immediate release of the drug into an absorption pool. In general,the term “extended release” as used herein includes controlled release,sustained release, and delayed release formulations.

By “pharmaceutically acceptable” is meant a material that is notbiologically or otherwise undesirable, i.e., the material may beincorporated into a pharmaceutical composition administered to a patientwithout causing any significant undesirable biological effects orinteracting in a deleterious manner with any of the other components ofthe composition in which it is contained. When the term“pharmaceutically acceptable” is used to refer to a pharmaceuticalcarrier or excipient, it is implied that the carrier or excipient hasmet the required standards of toxicological and manufacturing testing orthat it is included on the Inactive Ingredient Guide prepared by theU.S. Food and Drug administration.

The term “disorder” or “disease” used interchangeably herein, refers toany alteration in the state of the body or one of its organs and/ortissues, interrupting or disturbing the performance of organ functionand/or tissue function (e.g., causes organ dysfunction) and/or causing asymptom such as discomfort, dysfunction, distress, or even death to asubject afflicted with the disease.

The term “individual” refers to an animal, for example, a mammal andincludes, but is not limited to, human, bovine, horse, feline, canine,rodent, or primate. Preferably, the individual is a human.

The term “derivative” as used herein includes derivatives, analogs,prodrugs, and unnatural precursors.

The term “pharmaceutically acceptable salt” refers to a salt whichretains the biological effectiveness of the compound and which is notbiologically or otherwise undesirable.

Reference to “about” a value or parameter herein includes (anddescribes) variations that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X”.

As used herein and in the appended claims, the singular forms “a,” “or,”and “the” include plural referents unless the context clearly dictatesotherwise. It is understood that embodiments, aspects and variations ofthe invention described herein include “comprising,” “consisting” and/or“consisting essentially of” embodiments, aspects and variations.

Extended Release Formulations

Provided herein are extended release pharmaceutical formulationscomprising as the therapeutic agent one or more compounds in the sialicacid biosynthetic pathway or a derivative thereof or a pharmaceuticallyacceptable salt of the foregoing. In one embodiment, the extendedrelease pharmaceutical formulations comprise a therapeutic agent asdetailed herein and a polymer. An extended release formulationcomprising a therapeutic agent and a polymer may further comprise one ormore additional components, such as any one or more of a diluent, anexcipient, an antioxidant, a lubricant, a colorant, a binder, adisintegrant, and the like. It is understood that reference to anddescription of extended release pharmaceutical formulations comprisingone or more compounds in the sialic acid biosynthetic pathway or aderivative thereof below is exemplary and that this description appliesequally to and includes extended release pharmaceutical formulationscomprising any one or more compounds in the sialic acid biosyntheticpathway. It is also understood that reference to and description ofextended release pharmaceutical formulations comprising any one or morederivatives of compounds in the sialic acid biosynthetic pathway belowis exemplary and that this description applies equally to and includesextended release pharmaceutical formulations comprising any one or morederivatives, analogs, produgs, and/or unnatural precursor compounds inthe sialic acid biosynthetic pathway.

In one varation, the extended release formulation comprises sialic acid,or a pharmaceutically acceptable salt thereof, as the therapeutic agent.In another variation, the extended release formulation comprises MaNAc,or a pharmaceutically acceptable salt thereof, as the therapeutic agent.In another variation, the extended release formulation comprises sialicacid, or a pharmaceutically acceptable salt thereof and MaNAc, or apharmaceutically acceptable salt thereof, as the therapeutic agents. Inanother variation, the extended release formulation comprises a prodrugof one or more compounds in the sialic acid biosynthetic pathway such asa prodrug of sialic acid, or a pharmaceutically acceptable salt thereof,as the therapeutic agent. In another variation, the extended releaseformulation comrpises sialic acid, or a pharmaceutically acceptable saltthereof, as the therapeutic agent and further comprises a hydocolloidpolymer, an anionic, pH-dependent gel forming co-polymer and a waterswellable, pH independent polymer and optionally further comprises alubricant and/or an excipient. In a particular variation, the extendedrelease formulation comprises sialic acid, or a pharmaceuticallyacceptable salt thereof, carrageenan, sodium alginate and eitherhypromellose or polyethylene oxide. In a further variation, the extendedrelease formulation comprises sialic acid, or a pharmaceuticallyacceptable salt thereof, carrageenan, sodium alginate, eitherhypromellose or polyethylene oxide, magnesium strearate andmicrocrystalline collulose and colloiddal silicon dioxide. In oneaspect, the extended release formulation is a formulation of Table E. Inyet another aspect, the extended release formulation is a formulation ofTable 8.

Therapeutic Agent

It is believed that administration of sialic acid or a compound in thesialic acid biosynthetic pathway, or a derivative thereof, or apharmaceutically acceptable salt of any of the foregoing, may beadministered as a therapeutic agent (e.g., as substrate replacement) toan individual who has or is suspected of having a sialic acid deficiencydisorder. Extended release formulations comprising such compounds, orpharmaceutically acceptable salts thereof, as the therapeutic agent areprovided herein. In one aspect, the sialic acid or a compound in thesialic acid biosynthetic pathway, or a derivative thereof, or apharmaceutically acceptable salt of any of the foregoing, is sialic acidor a pharmaceutically acceptable salt thereof. In one aspect, any of theextended release formulations detailed herein may comprise an effectiveamount of a therapeutic agent, such as an effective amount of sialicacid or a pharmaceutically acceptable salt thereof.

A compound in the sialic acid biosynthetic pathway or a derivativethereof in one variation is a compound, or pharmaceutically acceptablesalt thereof, that is at or downstream from ManNAc in the sialic acidbiosynthetic pathway. In a particular variation, the therapeutic agentis a compound, or pharmaceutically acceptable salt thereof, that is ator downstream from ManNAc in the sialic acid biosynthetic pathway and isdepicted in FIG. 1.

A compound in the sialic acid biosynthetic pathway or a derivativethereof in another variation is a compound, or a pharmaceuticallyacceptable salt thereof, that is at or upstream from CMP-sialic acid inthe sialic acid biosynthetic pathway. In a particular variation, thetherapeutic agent is a compound, or a pharmaceutically acceptable saltthereof, that is at or upstream from CMP-sialic acid in the sialic acidbiosynthetic pathway and is depicted in FIG. 1. In one such variation,the compound in the sialic acid biosynthetic pathway or a derivativethereof does not include glucose or a pharmaceutically acceptable saltthereof.

In a particular variation, the compound in the sialic acid biosyntheticpathway or a derivative thereof in one variation is a compound, or apharmaceutically acceptable salt thereof, that is: (i) at or downstreamfrom ManNAc in the sialic acid biosynthetic pathway, and (ii) is at orupstream from CMP-sialic acid in the sialic acid biosynthetic pathway.In one such variation, the compound is a compound depicted in FIG. 1, ora pharmaceutically acceptable salt thereof.

A compound in the sialic acid biosynthetic pathway or derivative thereofincludes, but is not limited to, mannosamine, N-acetyl mannosamine(ManNAc), ManNac-6-phosphate (ManNAc-6-P), UDP-GlcNAc,N-acetylneuraminic acid (NeuAc), NeuAc-9-phosphate (NeuAc-9-P), sialicacid (i.e., 5-N-acetylneuraminic acid), CMP-sialic acid, and/orderivatives thereof or pharmaceutically acceptable salts of theforegoing.

In some embodiments, the one or more compounds in the sialic acidbiosynthetic pathway or derivative thereof include N-acetylneuraminicacid (NeuAc) or a derivative thereof. Structures of such NeuAc orderivatives thereof include, but are not limited to, those defined bythe formula below:

wherein each R₁, R₂, R₃, R₅, R₆, or R₇ is independently hydrogen, loweralkanoyl, carboxylate or lower alkyl; and R₄ is lower alkyl, loweralkanoylalkyl or lower alkyl alkanoyloxy.

In some embodiments, the one or more compounds in the sialic acidbiosynthetic pathway or derivative thereof include ManNAc or aderivative thereof. Structures of such ManNAc and derivatives thereofinclude, but are not limited to, those defined by the formula below:

Wherein each R₁, R₃, R₄, or R₅ is independently hydrogen, loweralkanoyl, carboxylate or lower alkyl; and R₂ is lower alkyl, loweralkanoylalkyl or lower alkyl alkanoyloxy.

The term lower alkyl refers to (C₁-C₆)alkyl. A lower alkyl includesmethyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl, pentyl,3-pentyl, hexyl as well as (C₃-C₆)cycloalkyl moieties (e.g.,cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl),(C₃-C₆)cycloalkyl(C₁-C₆)alkyl (e.g., cyclopropylmethyl,cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl,2-cyclopropylethyl, 2-cyclobutylethyl, 2-cyclopentylethyl, or2-cyclohexylethyl), (C₁-C₆)alkoxy (e.g., methoxy, ethoxy, propoxy,isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy, orhexyloxy) (C₂-C₆)alkenyl (e.g., vinyl, allyl, 1-propenyl, 2-propenyl,1-butenyl, 2-butenyl, 3-butenyl, 1,-pentenyl, 2-pentenyl, 3-pentenyl,4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, or 5-hexenyl),(C₂-C₆)alkynyl (e.g., ethynyl, 1-propynyl, 2-propynyl, 1-butynyl,2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl,1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, or 5-hexynyl),(C₁-C₆)alkanoyl (e.g., acetyl, propanoyl or butanoyl), halo(C₁-C₆)alkyl(e.g., iodomethyl, bromomethyl, chloromethyl, fluoromethyl,trifluoromethyl, 2-chloroethyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, orpentafluoroethyl), hydroxy(C₁-C₆)alkyl (e.g., hydroxymethyl,1-hydroxyethyl, 2-hydroxyethyl, 1-hydroxypropyl, 2-hydroxypropyl,3-hydroxypropyl, 1-hydroxy butyl, 4-hydroxybutyl, 1-hydroxypentyl,5-hydroxypentyl, 1-hydroxyhexyl, or 6-hydroxyhexyl),(C₁-C₆)alkoxycarbonyl (e.g., methoxycarbonyl, ethoxycarbonyl,propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, orhexyloxycarbonyl), (C₁-C₆)alkylthio (e.g., methylthio, ethylthio,propylthio, isopropylthio, butylthio, isobutylthio, pentylthio, orhexylthio), and/or (C₂-C₆)alkanoyloxy (e.g., acetoxy, propanoyloxy,butanoyloxy, isobutanoyloxy, pentanoyloxy, or hexanoyloxy).

In some embodiments, R₂ is methyl, and each of R₁, R₃, R₄, and R₅ ishydrogen. In some embodiments, the ManNAc or derivative thereof isN-acetyl mannosamine (ManNAc). In some embodiments, the ManNAc orderivative thereof is N-levulinoylmannosamine (ManLev) orN-azidoacetylmannosamine (ManNAz).

In one variation, the one or more compounds in the sialic acidbiosynthetic pathway or derivative thereof is an ester of a compound inthe sialic acid biosynthetic pathway. In one aspect, the one or morecompounds in the sialic acid biosynthetic pathway or derivative thereofis an ester of sialic acid or MaNAc. In a particular variation, the oneor more compounds in the sialic acid biosynthetic pathway or derivativethereof is an ester of sialic acid. In one aspect, the one or morecompounds in the sialic acid biosynthetic pathway or derivative thereofis a prodrug of sialic acid. See also WO 2010/131712, published Nov. 18,2010, for derivatives of compounds in the sialic acid biosyntheticpathway, which is incorporated herein by reference in its entirety andspecifically with respect to compounds (e.g., derivatives of compoundsin the sialic acid biosynthetic pathway) detailed therein.

In one aspect, a derivative of one or more compounds in the sialic acidbiosynthetic pathway (e.g., a derivative of sialic acid or MaNAc) is aneffective substrate replacement for sialic acid, such as in anindividual who has or is suspected of having a sialic acid deficiencydisorder. A derivative of one or more compounds in the sialic acidbiosynthetic pathway (e.g., a derivative of sialic acid or MaNAc), or anextended release formulation comprising a derivative of one or morecompounds in the sialic acid biosynthetic pathway (e.g., a derivative ofsialic acid or MaNAc) may exhibit any one or more of the followingcharacteristics: (i) capable of delivering to an individual in needthereof a therapeutically effective amount of one or more compounds inthe sialic acid pathway or derivatives thereof over a period of greaterthan about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, or 24 hours; (ii) capable of delivering toan individual in need thereof a substantially constant therapeuticallyeffective amount of one or more compounds in the sialic acid pathway orderivatives thereof over a period of greater than about any of 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, or 24 hours; (iii) capable of delivering to an individual in needthereof a therapeutically effective amount of one or more compounds inthe sialic acid pathway or derivatives thereof with a T_(max) of betweenabout any of 2-4 hours, 3-4 hours, 6-8 hours, 6-12 hours, 6-15 hours,12-18 hours, or 18-24 hours; (iv) capable of delivering to an individualin need thereof a therapeutically effective amount of one or morecompounds in the sialic acid pathway or derivatives thereof with aC_(max) of about 0.5-100 μg/mL; (v) capable of delivering to anindividual in need thereof a therapeutically effective amount of one ormore compounds in the sialic acid pathway or derivatives thereof with atrough level of about 0.1-20 μg/mL; (vi) capable of delivering to anindividual in need thereof a therapeutically effective amount of one ormore compounds in the sialic acid pathway or derivatives thereof withless than about any of 10%, 20%, 30%, 40%, 50%, 60%, or 70% excretedafter one hour; (vii) capable of delivering to an individual in needthereof between about any of 0.01-750 mg/kg/day, 0.5-500 mg/kg/day,1-250 mg/kg/day, 2.5-100 mg/kg/day, or 5-50 mg/kg/day of one or morecompounds in the sialic acid pathway or derivatives thereof or apharmaceutically acceptable salt of the foregoing; (viii) capable ofdelivering to an individual in need thereof between about any of0.01-750 mg/kg/day, 0.5-500 mg/kg/day, 1-250 mg/kg/day, 2.5-100mg/kg/day, or 5-50 mg/kg/day of one or more compounds in the sialic acidpathway or derivatives thereof or a pharmaceutically acceptable salt ofthe foregoing; (ix) has an absolute bioavailability of about 1 to about50%; (x) has a bioavailability based on sialic acid levels in the urineof about 0.5 to about 100%; and (xi) has a mean residence time (MRT) ofat least about 3.5 hours.

In some embodiments, the one or more compounds in the sialic acidbiosynthetic pathway or derivative thereof include sialic acid or aderivative thereof. In some embodiments, the sialic acid or derivativethereof is sialic acid. In some embodiments, the sialic acid orderivative thereof is a sialic acid analog such as N-levulinoyl sialicacid (SiaLev) or N-azidoacetyl sialic acid (SiaNAz). In someembodiments, the sialic acid is bound as a glycoconjugate. In someembodiments, the sialic acid or derivative thereof is an unnaturalprecursor such as sialylactose.

In some embodiments, the extended release formulation comprises aboutany of one, two, three, or four compounds in the sialic acidbiosynthetic pathway or a derivative thereof. In some embodiments, theextended release formulation comprises two compounds in the sialic acidbiosynthetic pathway or a derivative thereof. Therefore, for example,the extended release formulation may include ManNAc or a derivativethereof and sialic acid or a derivative thereof. More particularly, theextended release formulation may include ManNAc and sialic acid.

In embodiments of any of the extended release formulations, the amountof one or more compounds in the sialic acid biosynthetic pathway orderivative thereof in the extended release formulation is an amounteffective to increase sialic acid production and/or increase sialylation(e.g., maximal restoration of sialylation).

The ratio of the two or more compounds in the sialic acid biosyntheticpathway or derivative thereof, in some embodiments, is a ratio whichminimizes feedback inhibition of the sialic acid biosynthetic pathway.In some embodiments, the ratio of the two or more compounds in thesialic acid biosynthetic pathway or derivative thereof is a ratio whichallows efficient delivery of the two or more compounds in the sialicacid biosynthetic pathway or derivative thereof to muscle cells. In someembodiments, the ratio of the two or more compounds in the sialic acidbiosynthetic pathway or derivative thereof is a ratio which minimizesfeedback inhibition of the sialic acid biosynthetic pathway and allowsefficient delivery of the two or more compounds in the sialic acidbiosynthetic pathway or derivative thereof to muscle cells. In someembodiments, the two or more compounds in the sialic acid biosyntheticpathway or derivative there of are ManNAc or a derivative thereof andsialic acid or a derivative thereof. For example, in some embodiments,the ratio of ManNAc and sialic acid is a ratio which minimizes feedbackinhibition of the sialic acid biosynthetic pathway and allows efficientdelivery of ManNAc and/or sialic acid to muscle cells. The combinationmay optimally spread out the replacement of intermediates, enhancingoptimal distribution to all cell types with different metabolisms.Methods of testing restoration of sialylation and determining the bestratio of the two or more compounds in the sialic acid biosyntheticpathway or derivative thereof using in vitro HIBM muscle cells are knownin the art. See e.g., Noguchi S. et al., J. Bio. Chem. 279(12):11402-7(2004). This may involve evaluating muscle derived proteins for optimalsialylation such as soluble forms of neural cell adhesion molecule(NCAM) (Ricci et al., Neurology 66:755-758 (2006), evaluating sialicmetabolite or CMP-sialic acid levels in tissue samples, or assessingsialylated proteins on the surface of muscle or other cells. Noguchi S.et al., J. Bio. Chem. 279(12):11402-7 (2004).

In embodiments in which the extended release formulation comprises twocompounds in the sialic acid biosynthetic pathway or a derivativethereof, the two compounds in the extended release formulation may bepresent in a weight to weight percentage of between about any of5%-95%:95%-5%, 5%-50%:95%-50%, or 10%-40%:90%-60%. The two compounds inthe extended release formulation may be present in a weight to weightpercentage of about any of 90%:10%, 80%:20%, 70%:30%, 60%:40%, 50%:50%,40%:60%, 30%:70%, 20%:80%, or 10%:90%. In some embodiments, the twocompounds in the extended release formulation are in a weight to weightpercent of about 50%:50%. In some embodiments, the one or more compoundsin the sialic acid pathway or derivatives thereof include ManNAc or aderivative thereof and/or sialic acid or a derivative thereof.Therefore, for example, the extended release formulation may includeManNAc and sialic acid wherein the weight to weight percentage of ManNActo sialic acid is about any of 90%:10%, 80%:20%, 70%:30%, 60%:40%,50%:50%, 40%:60%, 30%:70%, 20%:80%, or 10%:90%.

Polymer

The extended release formulations comprising one or more compounds inthe sialic acid biosynthetic pathway or a derivative thereof asdescribed herein may include one or more polymers. The polymer may be anatural polymer (e.g., polysaccharide or protein), modified naturalpolymer, and/or synthetic polymer. The polymer may be, for example, ahydrophobic polymer, hydrophilic polymer, hydrogel, soluble polymer,biodegradable polymer, nonbiodegradable polymer, and/or mucoadhesivepolymer.

In some embodiments, the polymer is a hydrophobic polymer. Examples ofhydrophobic polymers include polyethylene, polyvinyl chloride, ethylcellulose or acrylate polymers and their copolymers.

In some embodiments, the polymer is a hydrophilic polymer. Examples ofhydrophilic polymers include a) cellulose derivatives such asmethylcellulose (MC), hydroxyethyl-cellulose,hydroxypropylmethyl-cellulose (HPMC), or sodium carboxymethylcellulose,b) noncellulose natural or semisynthetic polymers such as agar-agar,carob gum, alginates, molasses, polysaccharides of mannose andgalactose, or chitosan and modified starches and c) polymers of acrylicacid such as carbopol polymers.

In some embodiments, the polymer is a hydrogel. Examples of hydrogelsinclude, but are not limited to, polyhydroxyethyle methylacrylate(PHEMA), polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP),polyethylene oxide (PEO), or polyacrylamide (PA). In some embodiments,the hydrogel is polyethylene oxide (e.g., Polyox™ water soluble resin,Dow Chemical Company, Mich., USA).

In some embodiments, the polymer is a soluble polymer. Examples ofsoluble polymers include, but are not limited to, polyethylene glycol(PEG), PVA, PVP, or HPMC.

In some embodiments, the polymer is a biodegradable polymer. Examples ofbiodegradable polymers include, but are not limited to, polylactic acid(PLA), polyglycolic acid (PGA), poly(lactic/glycolic acid) (PLGA),polycaprolactone (PCL), polyanhydrides, or polyorthoesters.

In some embodiments, the polymer is a nonbiodegradable polymer. Examplesof nonbiodegradable polymers include, but are not limited to,polyethylene vinyl acetate, polydimethyl siloxane (PDS), polyetherurethane (PEU), polyvinyl chloride (PVC), cellulose acetate (CA), orethyl cellulose (EC).

In some embodiments, the polymer is a mucoadhesive polymer. Examples ofmucoadhesive polymers include, but are not limited to, polycarbophil,sodium carboxymethyl cellulose, polyacrylic acid, tragacanth, methylcellulose, pectin, natural gums, xanthan gum, guar gum, or karaya gum.

In some embodiments, the extended release pharmaceutical formulationincludes two polymers. In some embodiments, the polymer is notpolylactide. In some embodiments, the polymer is not a polylactidecopolymer such as PLGA.

In some embodiments, the extended release formulation comprises one ormore polymers selected from the group consisting of a) awater-swellable, pH independent polymer, b) a anionic, pH-dependent,gel-forming copolymer, c) a cationic polymer, and d) a hydrocolloidpolymer. In one varation, the extended release formulation comprisessialic acid, or a pharmaceutically acceptable salt thereof, as thetherapeutic agent. In another variation, the extended releaseformulation comprises MaNAc, or a pharmaceutically acceptable saltthereof, as the therapeutic agent. In another variation, the extendedrelease formulation comprises sialic acid, or a pharmaceuticallyacceptable salt thereof and MaNAc, or a pharmaceutically acceptable saltthereof, as the therapeutic agents. In another variation, the extendedrelease formulation comprises a prodrug of one or more compounds in thesialic acid biosynthetic pathway such as a prodrug of sialic acid, or apharmaceutically acceptable salt thereof, as the therapeutic agent. Inanother variation, the extended release formulation comrpises sialicacid, or a pharmaceutically acceptable salt thereof, as the therapeuticagent and further comprises a hydocolloid polymer, an anionic,pH-dependent gel forming co-polymer and a water swellable, pHindependent polymer and optionally further comprises a lubricant and/oran excipient. In a particular variation, the extended releaseformulation comprises sialic acid, or a pharmaceutically acceptable saltthereof, carrageenan, sodium alginate and either hypromellose orpolyethylene oxide. In a further variation, the extended releaseformulation comprises sialic acid, or a pharmaceutically acceptable saltthereof, carrageenan, sodium alginate, either hypromellose orpolyethylene oxide, magnesium strearate and microcrystalline colluloseand colloiddal silicon dioxide. In one aspect, the extended releaseformulation is a formulation of Table E. In yet another aspect, theextended release formulation is a formulation of Table 8.

Examples of a water-swellable, pH independent polymer include, but arenot limited to, carbohydrate-based polymers such as, for example,hypromellose (formerly known as the family of hydroxypropylmethylcellulose), hydroxypropyl ethyl celluloses, hydroxypropylcellulose, hydroxyethyl cellulose, methyl cellulose or otherconstituents Grades of these hypromellose copolymers typically used withthe present invention include the E and K series such as for example,Dow Chemical Company's (Midland, Mich. USA) or Aqualon's (with a NorthAmerican presence in Wilmington, Del.) E4M, E10M, K100LV, K4M, K15M,K25M, K100M, K200M and mixtures of various molecular weights and grades.Grades of hydroxyethyl cellulose include, for example, Aqualon'sNatrasol polymers HHX (mol. Wt. 1,300,000), HX (mol. wt. 1,000,000), H(mol. wt. 1,000,000), M (mol. wt. 720,000 and G (mol. wt. 1,150,000),and mixtures thereof. Grades of hydroxypropyl cellulose include, forexample, Aqualon's HPC polymers MF and MXF (mol. wt. 580,000) and KF andHXF (mol. wt. 1,150,000), and mixtures thereof. Grades and ethylcellulose include, for example, Dow Chemical Company's Ethocel polymers7FP, 10FP and 100FP and Aqualon's polymers T10EC, N7, N10, N17, N22,N50, N100 and N200, and mixtures thereof. In some embodiments, thewater-swellable, pH independent polymer is hypromellose (e.g.,hypromellose Type 2208). In some embodiments, the water-swellable, pHindependent polymer is Methocel® (e.g., Methocel® K100MPremium CR,Colorcon).

Examples of anionic, pH-dependent, gel-forming copolymer include, butare not limited to, mono-valent alginate salt such as sodium, potassiumor ammonium alginate salts, or combinations thereof, and sodiumcarboxymethyl cellulose and the like, or mixtures of one or morealginate salt and carboxymethyl cellulose and the like. In someembodiments, the anionic, pH-dependent, gel-forming copolymer is sodiumalginate (e.g., Protanal®, FMC BioPolymer).

Examples of a cationic polymer include, for example, chitosan or aderivative thereof including, for example, trimethylchitosan andquartermised chitosan, and chitosan-derived materials including, forexample, those taught in U.S. Pat. No. 5,747,475. Either high or lowmolecular weight chitosan products can be used in the pharmaceuticalformulations of the present invention and are readily available inpharmaceutical grade from suppliers located world-wide.

The hydrocolloid polymer used in the formulations of the presentinvention can be carrageenan. Carrageenans are available as iota, kappaand lambda carrageenans, with iota being used most frequently used andlambda being used least frequently. Various salt forms of carrageenansare also available including, for example sodium carrageenan. Typicallyused grades of iota carrageenan include, without limitation, carrageenanNF AEP brand colloids (Hadley, N.Y. USA) FD433 (1% viscosity; 300-400cps) and FD384 (1% viscosity; about 100 cps). Viscosity of othercarrageenan products ranges from about 50 to about 4000 cps. In someembodiments, the carrageenan is lambda carrageenan (e.g., ViscarinGP-209, FMC BioPolymer). In some embodiments, the carrageenan has aviscosity of about 1500-2000 cPs. In some embodiments, the carrageenanhas a viscosity of about 1600 cPs.

The formulation and polymers useful in the extended release formulationare further described in U.S. Patent Application 2010/0160363, publishedon Jun. 24, 2010, and U.S. Patent Application 2010/0159001, publishedJun. 24, 2010, which are incorporated herein by reference in theirentireties and specifically with respect to the polymers providedtherein.

In some embodiments, the extended release formulation comprises awater-swellable, pH independent polymer (e.g., hypromellose). In someembodiments, the extended release formulation further comprises ananionic, pH-dependent, gel-forming copolymer (e.g., an alginate salt).In some embodiments, the extended release formulation further comprisesa hydrocolloid polymer (e.g., carrageenan). In some embodiments, theextended release formulation comprises a water-swellable, pH independentpolymer (e.g. hypromellose), an anionic, pH-dependent, gel-formingcopolymer (e.g., an alginate salt) and a hydrocolloid polymer (e.g., acarrageenan). In some embodiments, the extended release formulationcomprises hypromellose (e.g. hypromellose Type 2208 or Methocel K100M),sodium alginate (e.g. Protanal) and a lamda carrageenan (e.g. ViscarinGP-209). In one varation, the extended release formulation comprisessialic acid, or a pharmaceutically acceptable salt thereof, as thetherapeutic agent. In another variation, the extended releaseformulation comprises MaNAc, or a pharmaceutically acceptable saltthereof, as the therapeutic agent. In another variation, the extendedrelease formulation comprises sialic acid, or a pharmaceuticallyacceptable salt thereof and MaNAc, or a pharmaceutically acceptable saltthereof, as the therapeutic agents. In another variation, the extendedrelease formulation comprises a prodrug of one or more compounds in thesialic acid biosynthetic pathway such as a prodrug of sialic acid, or apharmaceutically acceptable salt thereof, as the therapeutic agent. Inanother variation, the extended release formulation comrpises sialicacid, or a pharmaceutically acceptable salt thereof, as the therapeuticagent and further comprises a hydocolloid polymer, an anionic,pH-dependent gel forming co-polymer and a water swellable, pHindependent polymer and optionally further comprises a lubricant and/oran excipient. In a particular variation, the extended releaseformulation comprises sialic acid, or a pharmaceutically acceptable saltthereof, carrageenan, sodium alginate and either hypromellose orpolyethylene oxide. In a further variation, the extended releaseformulation comprises sialic acid, or a pharmaceutically acceptable saltthereof, carrageenan, sodium alginate, either hypromellose orpolyethylene oxide, magnesium strearate and microcrystalline colluloseand colloiddal silicon dioxide. In one aspect, the extended releaseformulation is a formulation of Table E. In yet another aspect, theextended release formulation is a formulation of Table 8.

In some embodiments, the extended release formulation comprises ahydrogel (e.g., a polyethylene oxide). In some embodiments, the extendedrelease formulation further comprises an anionic, pH-dependent,gel-forming copolymer (e.g. an alginate salt). In some embodiments, theextended release formulation further comprises a hydrocholloid polymer(e.g., carrageenan). In some embodiments, the extended releaseformulation comprises a hydrogel (e.g., a polyethylene oxide), ananionic, pH-dependent, gel-forming copolymer (e.g., an alginate salt)and a hydrocholloid polymer (e.g., a carrageenan). In some embodiments,the extended release formulation comprises polyethylene oxide (e.g.Polyox WSR), sodium alginate (e.g. Protanal) and a lamda carrageenan(e.g. Viscarin GP-209). In one varation, the extended releaseformulation comprises sialic acid, or a pharmaceutically acceptable saltthereof, as the therapeutic agent. In another variation, the extendedrelease formulation comprises MaNAc, or a pharmaceutically acceptablesalt thereof, as the therapeutic agent. In another variation, theextended release formulation comprises sialic acid, or apharmaceutically acceptable salt thereof and MaNAc, or apharmaceutically acceptable salt thereof, as the therapeutic agents. Inanother variation, the extended release formulation comprises a prodrugof one or more compounds in the sialic acid biosynthetic pathway such asa prodrug of sialic acid, or a pharmaceutically acceptable salt thereof,as the therapeutic agent. In another variation, the extended releaseformulation comrpises sialic acid, or a pharmaceutically acceptable saltthereof, as the therapeutic agent and further comprises a hydocolloidpolymer, an anionic, pH-dependent gel forming co-polymer and a waterswellable, pH independent polymer and optionally further comprises alubricant and/or an excipient. In a particular variation, the extendedrelease formulation comprises sialic acid, or a pharmaceuticallyacceptable salt thereof, carrageenan, sodium alginate and eitherhypromellose or polyethylene oxide. In a further variation, the extendedrelease formulation comprises sialic acid, or a pharmaceuticallyacceptable salt thereof, carrageenan, sodium alginate, eitherhypromellose or polyethylene oxide, magnesium strearate andmicrocrystalline collulose and colloiddal silicon dioxide. In oneaspect, the extended release formulation is a formulation of Table E. Inyet another aspect, the extended release formulation is a formulation ofTable 8.

In one variation, the extended release formulation comprises: (i) ahydrocolloid polymer; (ii) an anionic, pH-dependent, gel formingco-polymer, and (iii) either a water-swellable, pH independent polymeror a hydrogel. In one varation, the extended release formulationcomprises sialic acid, or a pharmaceutically acceptable salt thereof, asthe therapeutic agent. In another variation, the extended releaseformulation comprises MaNAc, or a pharmaceutically acceptable saltthereof, as the therapeutic agent. In another variation, the extendedrelease formulation comprises sialic acid, or a pharmaceuticallyacceptable salt thereof and MaNAc, or a pharmaceutically acceptable saltthereof, as the therapeutic agents. In another variation, the extendedrelease formulation comprises a prodrug of one or more compounds in thesialic acid biosynthetic pathway such as a prodrug of sialic acid, or apharmaceutically acceptable salt thereof, as the therapeutic agent. Inanother variation, the extended release formulation comrpises sialicacid, or a pharmaceutically acceptable salt thereof, as the therapeuticagent and further comprises a hydocolloid polymer, an anionic,pH-dependent gel forming co-polymer and a water swellable, pHindependent polymer and optionally further comprises a lubricant and/oran excipient. In a particular variation, the extended releaseformulation comprises sialic acid, or a pharmaceutically acceptable saltthereof, carrageenan, sodium alginate and either hypromellose orpolyethylene oxide. In a further variation, the extended releaseformulation comprises sialic acid, or a pharmaceutically acceptable saltthereof, carrageenan, sodium alginate, either hypromellose orpolyethylene oxide, magnesium strearate and microcrystalline colluloseand colloiddal silicon dioxide. In one aspect, the extended releaseformulation is a formulation of Table E. In yet another aspect, theextended release formulation is a formulation of Table 8. An extendedrelease formulation in one variation comprises a therapeutic agent asdetailed herein (e.g., sialic acid) and (i) a hydrocolloid polymer; (ii)an anionic, pH-dependent, gel forming co-polymer, and (iii) either awater-swellable, pH independent polymer or a hydrogel. Exemplaryextended release formulations include those listed in Table A, where itis understood that an extended release formulation may comprise any ofthe listed therapeutic agents in combination with at least one of any ofpolymers 1, 2, 3A or 3B the same as if each and every combination oftherapeutic agent and polymer or combination of polymers werespecifically and individually listed. Although particular formulationsmay comprise a therapeutic agent of Table A and any one or more of apolymer selected from Polymers 1, 2 and 3 (A and/or B) of Table A, in aparticular variation, an extended release formulation comprises atherapeutic agent of Table A, a polymer 1 of Table A, a polymer 2 ofTable A and either a polymer 3A of Table A or a polymer 3B of Table Athe same as if each and every combination of therapeutic agent andpolymer combination were specifically and individually listed. Forexample, it is understood that in one aspect, an extended releaseformulation comprises sialic acid, carrageenan (e.g., a lambdacarrageenan such as Viscarin GP-209), an alginate salt (e.g., sodiumalginate such as Protanal® LF 120M), and either (i) hypromellose (e.g.,hypromellose Type 2208) or (ii) polyethylene oxide (e.g., Polyox), or apharmaceutically acceptable salt of any of the foregoing.

TABLE A Exemplary Components for use in Extended Realease Formulations.Therapeutic mannosamine, N-acetyl mannosamine (ManNAc),ManNac-6-phosphate Agent (ManNAc-6-P), UDP-GlcNAc, N-acetylneuraminicacid (NeuAc), NeuAc- (A compound in 9-phosphate (NeuAc-9-P), sialic acid(i.e., 5-N-acetylneuraminic acid), the sialic acid CMP-sialic acid,and/or derivatives thereof or pharmaceutically acceptable biosyntheticsalts of the foregoing. pathway or derivative thereof or salt of any ofthe foregoing) Polymer 1 Carrageenan (e.g., iota, kappa or lambdacarrageenan, or a salt thereof, such (Hydrocolloid as Viscarin GP-209,FMC BioPolymer) Polymer) Polymer 2 Alginate or salt thereof (e.g.,sodium, potassium or ammonium alginate salt (Anionic, pH- such asProtanal ®, FMC BioPolymer), carboxymethyl cellulose or salt dependent,gel thereof (e.g., sodium carboxymethyl cellulose). forming co- polymer)Polymer 3A Hypromellose (e.g., hypromellose Type 2208, E and K seriessuch as for (Water-swellable, example, Dow Chemical Company's (Midland,Mich. USA) or Aqualon's pH independent (with a North American presencein Wilmington, Del.) E4M, E10M, polymer) K100LV, K4M, K15M, K25M, K100M,K200M and mixtures of various molecular weights and grades);hydroxypropyl ethyl cellulose (Ethocel polymers 7FP, 10FP and 100FP andAqualon's polymers T10EC, N7, N10, N17, N22, N50, N100 and N200, andmixtures thereof), hydroxypropyl cellulose (Aqualon's HPC polymers MFand MXF (mol. wt. 580,000) and KF and HXF (mol. wt. 1,150,000), andmixtures thereof); hydroxyethyl cellulose (e.g., Aqualon's Natrasolpolymers HHX (mol. Wt. 1,300,000), HX (mol. wt. 1,000,000), H (mol. wt.1,000,000), M (mol. wt. 720,000 and G (mol. wt. 1,150,000), and mixturesthereof); methyl cellulose.. Polymer 3B Polyhydroxyethyle methylacrylate(PHEMA), polyvinyl alcohol (PVA), (Hydrogel) polyvinyl pyrrolidone(PVP), polyethylene oxide (PEO), polyacrylamide (PA), polyethylene oxide(e.g., Polyox ™ water soluble resin, Dow Chemical Company, Mich., USA).

In one variation, an extended release formulation comprises atherapeutic agent of Table A, a polymer 1 of Table A, a polymer 2 ofTable A and either a polymer 3A or a polymer 3B of Table A, wherein thecomposition comprises the therapeutic agent and polymers in any one ofthe weight percent ranges depicted in Table B.

TABLE B Exemplary Weight Percent of Certain Components for Use inExtended Release Formulations. Formulation Component Weight Percent ofComponent in Formulation Therapeutic Agent (A From about 20 to about 80;from about 20 to about 60; from about compound in the sialic acid 20 toabout 50; from about 20 to about 40; from about 20 to about biosyntheticpathway or 30; from about 15 to about 60; from about 15 to about 50;from derivative thereof or salt of about 15 to about 40; from about 25to about 60; from about 25 to any of the foregoing) about 50; from 25 toabout 40; from about 25 to about 30; from about 30 to about 60; fromabout 30 to about 50; from about 30 to about 45; from about 30 to about40; from about 35 to about 60; from about 35 to about 50; from about 35to about 45; from about 40 to about 45; about any of 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50. Polymer 1 (HydrocolloidFrom about 1 to about 10; from about 1 to about 5; from about 3 toPolymer) about 8; from about 4 to about 6; about any of 1, 2, 3, 4, 5,6, 7, 8, 9 and 10. Polymer 2 (Anionic, pH- From about 15 to about 30;from about 15 to about 25; from about dependent, gel forming 15 to about20; from about 20 to about 30; from about 20 to about co-polymer) 25;from about 2o to about 23; about any of 15, 16, 17, 18, 19, 20, 21, 22,23, 24 and 25. Polymer 3A. (Water- From about 20 to about 50; from about20 to about 40, from about swellable, pH independent 20 to about 30;from about 20 to about 25; from about 25 to about polymer) 30; fromabout 22 to about 27; about any of 20, 21, 22, 23, 24, 25, Or 26, 27,28, 29 and 30. Polymer 3B. (Hydrogel)

In another variation, the extended release formulation comprises atherapeutic agent (a compound in the sialic acid biosynthetic pathway orderivative thereof or salt of any of the foregoing, such as any of thecompounds detailed herein, including in Table A) and a polymer, whereinthe polymer comprises: (i) a hydrocolloid polymer; (ii) an anionic,pH-dependent, gel forming co-polymer, and (iii) either awater-swellable, pH independent polymer or a hydrogel, and wherein theweight percent ratio of polymers (i):(ii):(iii) is about 1:5:5 or about1:5:6.

The combination of (i) a hydrocolloid polymer; (ii) an anionic,pH-dependent, gel forming co-polymer, and (iii) either awater-swellable, pH independent polymer or a hydrogel is believed toprovide a unique combination that is particularly advantageous for thepreparation of oral dosage forms in that the combination results in anyone or more of the following features: (i) provides a robust formulation(e.g., for tablet formulation); (i) is pH independent; and (iii) lendsitself to granulation without affecting dissolution profile.

Further descriptions of extended release formulations and formulationcomponents are found throughout and below.

It is understood that reference to relative weight percentages assumesthat the combined total weight percentages of all components in theformulation add up to 100. It is further understood that relative weightpercentages of one or more components may be adjusted upwards ordownwards such that the weight percent of the components in thecomposition combine to a total of 100. In one aspect, the weightpercentages detailed herein refer to the weight percentages of aformulation blend (e.g., prior to formulation into a unit dosage amountsuch as a tablet, which may be further modified, e.g., by the additionof a tablet coating). In another aspect, the weight percentages detailedherein refer to the weight percentages of a unit dosage of aformulation, in which the formulation is in a form and/or packaged foradministration to an individual (e.g., a tablet that has a coating).

The polymer may be present in the extended release formulation in anamount ranging from 5 to 40 parts by weight, from 10 to 20 parts byweight, relative to 100 parts by weight of the one or more compounds inthe sialic acid pathway or derivatives thereof. In some embodiments, theone or more compounds in the sialic acid pathway or derivatives thereofin such formulations includes from about 0.1 to 99.9% by weight of theformulation. In some embodiments, the one or more compounds in thesialic acid pathway or derivatives thereof in such formulations includesabout any of between 20%-30%, 30%-40%, 40%-50%, or 20%-50%. In someembodiments, the extend release formulation includes about any ofbetween 40%-50%, 50%-60%, 60%-70%, or 50% to 70% by weight of polymer.

In some embodiments, the drug load of the one or more compounds in thesialic acid pathway or derivatives thereof in the extended releaseformulation comprises about 20% to 80% w/w. In some embodiments, thedrug load of the one or more compounds in the sialic acid pathway orderivatives thereof in the extended release formulation comprises aboutany one of 20% to 60% w/w, 20%-50% w/w, 20%-40% w/w, 15%-60% w/w,15%-50% w/w, 15%-40% w/w, 25%-0.60% w/w, 25%-50% w/w, 25%-40% w/w,30%-60% w/w, 30%-50% w/w, 30%-45% w/w, 35%-60% w/w, 35%-50% w/w, or35%-45% w/w. In some embodiments, the drug load of the one or morecompounds in the sialic acid pathway or derivatives thereof in theextended release formulation comprises at least about any one of 25%w/w, 30% w/w, 35% w/w, 40% w/w, 45% w/w, or 50% w/w. In someembodiments, the drug load of the one or more compounds in the sialicacid pathway or derivatives thereof in the extended release formulationcomprises about 33% w/w. In some embodiments, the drug load of the oneor more compounds in the sialic acid pathway or derivatives thereof inthe extended release formulation comprises about 43% w/w

In some embodiments, the extended release formulation comprises about 20to about 50 or about 20 to about 40 or about 20 to about 30% w/w of awater-swellable, pH independent polymer (e.g., hypromellose). In someembodiments, the extended release formulation comprises about 25% w/w ofa water-swellable, pH independent polymer (e.g., hypromellose). In someembodiments, the extended release formulation further comprises about20-25% w/w of an anionic, pH-dependent, gel-forming copolymer (e.g., analginate salt). In some embodiments, the extended release formulationfurther comprises about 21% w/w of an anionic, pH-dependent, gel-formingcopolymer (e.g., an alginate salt). In some embodiments, the extendedrelease formulation further comprises about 1-5% w/w of a hydrocolloidpolymer (e.g., carrageenan). In some embodiments, the extended releaseformulation further comprises about 4% w/w of a hydrocolloid polymer(e.g., carrageenan). In some embodiments, the extended releaseformulation comprises about 20-30% w/w of a water-swellable, pHindependent polymer (e.g. hypromellose), about 20-25% w/w of an anionic,pH-dependent, gel-forming copolymer (e.g., an alginate salt) and about1-5% w/w of a hydrocolloid polymer (e.g., a carrageenan). In someembodiments, the extended release formulation comprises about 20-30% w/whypromellose (e.g. hypromellose Type 2208 or Methocel K100M), about20-25% w/w sodium alginate (e.g. Protanal) and about 1-5% w/w lamdacarrageenan (e.g. Viscarin GP-209). In some embodiments, the extendedrelease formulation comprises about 25% w/w of a water-swellable, pHindependent polymer (e.g. hypromellose), about 21% w/w of an anionic,pH-dependent, gel-forming copolymer (e.g., an alginate salt) and about4% w/w of a hydrocolloid polymer (e.g., a carrageenan). In someembodiments, the extended release formulation comprises about 25% w/whypromellose (e.g. hypromellose Type 2208 or Methocel K100M), about 21%w/w sodium alginate (e.g. Protanal) and about 4% w/w lamda carrageenan(e.g. Viscarin GP-209).

In some embodiments, the extended release formulation comprises about 20to about 50 or about 20 to about 40 or about 20 to about 20-30% w/w of ahydrogel (e.g., a polyethylene oxide, Polyox WSR). In some embodiments,the extended release formulation comprises about 25% w/w of a hydrogel(e.g., a polyethylene oxide, Polyox WSR). In some embodiments, theextended release formulation further comprises about 20-25% w/w of ananionic, pH-dependent, gel-forming copolymer (e.g., an alginate salt).In some embodiments, the extended release formulation further comprisesabout 21% w/w of an anionic, pH-dependent, gel-forming copolymer (e.g.,an alginate salt). In some embodiments, the extended release formulationfurther comprises about 1-5% w/w of a hydrocolloid polymer (e.g.,carrageenan). In some embodiments, the extended release formulationfurther comprises about 4% w/w of a hydrocolloid polymer (e.g.,carrageenan). In some embodiments, the extended release formulationcomprises about 20-30% w/w of a hydrogel (e.g., a polyethylene oxide),about 20-25% w/w of an anionic, pH-dependent, gel-forming copolymer(e.g., an alginate salt) and about 1-5% w/w of a hydrocolloid polymer(e.g., a carrageenan). In some embodiments, the extended releaseformulation comprises about 20-30% w/w polyethylene oxide (e.g. PolyoxWSR), about 20-25% w/w sodium alginate (e.g. Protanal) and about 1-5%w/w lamda carrageenan (e.g. Viscarin GP-209). In some embodiments, theextended release formulation comprises about 25% w/w of a hydrogel(e.g., a polyethylene oxide), about 21% w/w of an anionic, pH-dependent,gel-forming copolymer (e.g., an alginate salt) and about 4% w/w of ahydrocolloid polymer (e.g., a carrageenan). In some embodiments, theextended release formulation comprises about 25% w/w polyethylene oxide(e.g. Polyox WSR), about 21% w/w sodium alginate (e.g. Protanal) andabout 4% w/w lamda carrageenan (e.g. Viscarin GP-209).

Additional Formulation Components

The extended release pharmaceutical formulations comprising one or morecompounds in the sialic acid biosynthetic pathway or derivative thereofas described herein may further comprise a diluent, an excipient, anantioxidant, a lubricant, a colorant, a binder, a disintegrant, and thelike. It is understood that any of the extended release formulationsdetailed herein, including but not limited to those listed under theheading “Extended Release Formulations” (e.g., any formulation of TablesA or B) may further comprise a diluent, an excipient, an antioxidant, alubricant, a colorant, a binder, a disintegrant, and the like asdetailed herein the same as if each and every extended releaseformulation further comprising such a component were specifically andindividually listed. In one varation, the extended release formulationcomprises sialic acid, or a pharmaceutically acceptable salt thereof, asthe therapeutic agent. In another variation, the extended releaseformulation comprises MaNAc, or a pharmaceutically acceptable saltthereof, as the therapeutic agent. In another variation, the extendedrelease formulation comprises sialic acid, or a pharmaceuticallyacceptable salt thereof and MaNAc, or a pharmaceutically acceptable saltthereof, as the therapeutic agents. In another variation, the extendedrelease formulation comprises a prodrug of one or more compounds in thesialic acid biosynthetic pathway such as a prodrug of sialic acid, or apharmaceutically acceptable salt thereof, as the therapeutic agent. Inanother variation, the extended release formulation comrpises sialicacid, or a pharmaceutically acceptable salt thereof, as the therapeuticagent and further comprises a hydocolloid polymer, an anionic,pH-dependent gel forming co-polymer and a water swellable, pHindependent polymer and optionally further comprises a lubricant and/oran excipient. In a particular variation, the extended releaseformulation comprises sialic acid, or a pharmaceutically acceptable saltthereof, carrageenan, sodium alginate and either hypromellose orpolyethylene oxide. In a further variation, the extended releaseformulation comprises sialic acid, or a pharmaceutically acceptable saltthereof, carrageenan, sodium alginate, either hypromellose orpolyethylene oxide, magnesium strearate and microcrystalline colluloseand colloiddal silicon dioxide. In one aspect, the extended releaseformulation is a formulation of Table E. In yet another aspect, theextended release formulation is a formulation of Table 8.

The diluent is selected so as not to affect the biological activity ofthe combination. Examples of such diluents are distilled water, bufferedwater, physiological saline, PBS, Ringer's solution, dextrose solution,and Hank's solution. The pharmaceutical formulations can also includeadditional substances to approximate physiological conditions, such aspH adjusting and buffering agents, toxicity adjusting agents, wettingagents and detergents. The pharmaceutical formulations can also includeany of a variety of stabilizing agents. Further guidance regardingpharmaceutical formulations that are suitable for various types ofadministration can be found in Remington: The Science and Practice ofPharmacy, 20th Edition. Baltimore, Md.: Lippincott Williams & Wilkins,2000.

The excipient may be selected from the group consisting of lactose,microcrystalline cellulose, corn starch, potato starch, wheat starch,sucrose, D-mannitol, precipitated calcium carbonate, dextrin,pre-gelatinized starch, and combinations thereof. The excipient, ifpresent, may be contained in an amount of about 10 to about 90 parts byweight based on the total weight of the tablet. In some embodiments, theextend release formulation includes about any of between 40%-50%,50%-60%, 60%-70%, or 50% to 70% by weight of excipient. In someembodiments, the excipient is microcrystalline cellulose. In someembodiments, the excipient is microcrystalline cellulose and colloidalsilicon dioxide (e.g., ProSolv® SMCC HD90). In some embodiments, theextended release formulation comprises about 1-10% w/w ofmicrocrystalline cellulose and colloidal silicon dioxide (e.g., ProSolv®SMCC HD90). In some embodiments, the extended release formulationcomprises about 5% w/w of microcrystalline cellulose and colloidalsilicon dioxide (e.g., ProSolv® SMCC HD90).

The binder may be selected from the group consisting ofhydroxypropylcellulose, direct tabletted microcrystalline cellulose,HPMC, MC, hydroxyethylcellulose, hydroxymethylcellulose, carboxymethylcellulose, and other cellulose derivative, PVP, PVA, paste, arabic gum,dextrin, gelatin, alginates, and combinations thereof. The binder, ifpresent, may be used in an amount of about 2 to about 60 parts by weightbased on the total weight of the tablet.

The disintegrant may be selected from the group consisting of sodiumstarch glycolate, crosspovidone, cross carmellose sodium,low-substituted hydroxypropylcellulose, starch, carboxymethylcellulosecalcium, calcium carbonate, sodium bicarbonate, and combinationsthereof. The disintegrant, if present, may be contained in an amount ofabout 0.1 to about 32 parts by weight based on the total weight of thetablet composition.

The lubricant may be selected from the group consisting of magnesiumstearate, calcium stearate, talc, light anhydrous silicic acid, andsolid polyethyl glycols and combinations thereof. The lubricant, ifpresent, may be contained in an amount of about 0.1 to about 20 parts byweight based on the total weight of the tablet. In some embodiments, thelubricant is magnesium stearate (e.g., HyQual®). In some embodiments,the extended release formulation comprises about 0.1-1% w/w magnesiumstearate (e.g., HyQual®). In some embodiments, the extended releaseformulation comprises about 0.5% w/w magnesium stearate (e.g., HyQual®).

For the colorant, at least one species which can be selected fromtitanium dioxide, iron oxide, magnesium carbonate, calcium sulfate,magnesium oxide, magnesium hydroxide, aluminum lakes, for example, BlueNo. 1 Aluminum Lake, Red No. 40 Aluminum Lake, and the like can becontained in the tablet.

In another variation, an extended release formulation detailed herein(including but not limited to those listed under the heading “ExtendedRelease Formulations” (e.g., any formulation of Tables A and B) furthercomprises an excipient. In a particular variation, the excipientcomprises microcrystalline cellulose. In a further variation, theexcipient comprises microcrystalline cellulose and colloidal silicondioxide. In any such variations, the extended release formulationfurther comprising an excipient (e.g., an excipient comprisingmicrocrystalline cellulose and colloidal silicon dioxide) comprises theexcipient in about 1 to about 20 or about 1 to about 15 or about 1 toabout 10 or about 1 to about 5 or about 5 to about 20 or about 5 toabout 15 or about 5 to about 10 or about any one of 1, 2, 3, 4, 5, 6, 7,8, 9 or 10 weight percent. In one varation, the extended releaseformulation comprises sialic acid, or a pharmaceutically acceptable saltthereof, as the therapeutic agent. In another variation, the extendedrelease formulation comprises MaNAc, or a pharmaceutically acceptablesalt thereof, as the therapeutic agent. In another variation, theextended release formulation comprises sialic acid, or apharmaceutically acceptable salt thereof and MaNAc, or apharmaceutically acceptable salt thereof, as the therapeutic agents. Inanother variation, the extended release formulation comprises a prodrugof one or more compounds in the sialic acid biosynthetic pathway such asa prodrug of sialic acid, or a pharmaceutically acceptable salt thereof,as the therapeutic agent. In another variation, the extended releaseformulation comrpises sialic acid, or a pharmaceutically acceptable saltthereof, as the therapeutic agent and further comprises a hydocolloidpolymer, an anionic, pH-dependent gel forming co-polymer and a waterswellable, pH independent polymer and optionally further comprises alubricant and/or an excipient. In a particular variation, the extendedrelease formulation comprises sialic acid, or a pharmaceuticallyacceptable salt thereof, carrageenan, sodium alginate and eitherhypromellose or polyethylene oxide. In a further variation, the extendedrelease formulation comprises sialic acid, or a pharmaceuticallyacceptable salt thereof, carrageenan, sodium alginate, eitherhypromellose or polyethylene oxide, magnesium strearate andmicrocrystalline collulose and colloiddal silicon dioxide. In oneaspect, the extended release formulation is a formulation of Table E. Inyet another aspect, the extended release formulation is a formulation ofTable 8.

In another variation, an extended release formulation detailed herein(including but not limited to those listed under the heading “ExtendedRelease Formulations” (e.g., any formulation of Tables A and B) furthercomprises a lubricant. In a particular variation, the lubricantcomprises a stearate salt, such as magnesium stearate. In any suchvariations, the extended release formulation further comprising alubricant (e.g., a stearate salt such as magnesium stearate) comprisesthe lubricant in about 0.1 to about 2 or about 0.1 to about 1.5 or about0.1 to about 1.0 or about 0.01 to about 0.09-5 or about 0.1 to about 0.8or about 0.1 to about 0.7 or about 0.1 to about 0.6 or about 0.1 toabout 0.5 or about 0.2 to about 0.8 or about 0.3 to about 0.7 or about0.4 to about 0.6 or about any one of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9 or 1.0 weight percent. In one varation, the extended releaseformulation comprises sialic acid, or a pharmaceutically acceptable saltthereof, as the therapeutic agent. In another variation, the extendedrelease formulation comprises MaNAc, or a pharmaceutically acceptablesalt thereof, as the therapeutic agent. In another variation, theextended release formulation comprises sialic acid, or apharmaceutically acceptable salt thereof and MaNAc, or apharmaceutically acceptable salt thereof, as the therapeutic agents. Inanother variation, the extended release formulation comprises a prodrugof one or more compounds in the sialic acid biosynthetic pathway such asa prodrug of sialic acid, or a pharmaceutically acceptable salt thereof,as the therapeutic agent. In another variation, the extended releaseformulation comrpises sialic acid, or a pharmaceutically acceptable saltthereof, as the therapeutic agent and further comprises a hydocolloidpolymer, an anionic, pH-dependent gel forming co-polymer and a waterswellable, pH independent polymer and optionally further comprises alubricant and/or an excipient. In a particular variation, the extendedrelease formulation comprises sialic acid, or a pharmaceuticallyacceptable salt thereof, carrageenan, sodium alginate and eitherhypromellose or polyethylene oxide. In a further variation, the extendedrelease formulation comprises sialic acid, or a pharmaceuticallyacceptable salt thereof, carrageenan, sodium alginate, eitherhypromellose or polyethylene oxide, magnesium strearate andmicrocrystalline collulose and colloiddal silicon dioxide. In oneaspect, the extended release formulation is a formulation of Table E. Inyet another aspect, the extended release formulation is a formulation ofTable 8.

In a further variation, an extended release formulation detailed herein(including but not limited to those listed under the heading “ExtendedRelease Formulations” (e.g., any formulation of Tables A and B) furthercomprises both an excipient and a lubricant. In any such variation, theformulation further comprising both an excipient and a lubricantcomprises the excipient (e.g., an excipient comprising microcrystallinecellulose and colloidal silicon dioxide) in about 1 to about 20 or about1 to about 15 or about 1 to about 10 or about 1 to about 5 or about 5 toabout 20 or about 5 to about 15 or about 5 to about 10 or about any oneof 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 weight percent and comprises thelubricant (e.g., a stearate salt such as magnesium stearate) in about0.1 to about 2 or about 0.1 to about 1.5 or about 0.1 to about 1.0 orabout 0.01 to about 0.09-5 or about 0.1 to about 0.8 or about 0.1 toabout 0.7 or about 0.1 to about 0.6 or about 0.1 to about 0.5 or about0.2 to about 0.8 or about 0.3 to about 0.7 or about 0.4 to about 0.6 orabout any one of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0weight percent. In a further variation, the formulation furthercomprising both an excipient and a lubricant comprises the excipient(e.g., an excipient comprising microcrystalline cellulose and colloidalsilicon dioxide) in a weight percent ratio to lubricant (e.g., astearate salt such as magnesium stearate) of about any of 1:10 or 1:11or 1:9 or 1:10.5. In one varation, the extended release formulationcomprises sialic acid, or a pharmaceutically acceptable salt thereof, asthe therapeutic agent. In another variation, the extended releaseformulation comprises MaNAc, or a pharmaceutically acceptable saltthereof, as the therapeutic agent. In another variation, the extendedrelease formulation comprises sialic acid, or a pharmaceuticallyacceptable salt thereof and MaNAc, or a pharmaceutically acceptable saltthereof, as the therapeutic agents. In another variation, the extendedrelease formulation comprises a prodrug of one or more compounds in thesialic acid biosynthetic pathway such as a prodrug of sialic acid, or apharmaceutically acceptable salt thereof, as the therapeutic agent. Inanother variation, the extended release formulation comrpises sialicacid, or a pharmaceutically acceptable salt thereof, as the therapeuticagent and further comprises a hydocolloid polymer, an anionic,pH-dependent gel forming co-polymer and a water swellable, pHindependent polymer and optionally further comprises a lubricant and/oran excipient. In a particular variation, the extended releaseformulation comprises sialic acid, or a pharmaceutically acceptable saltthereof, carrageenan, sodium alginate and either hypromellose orpolyethylene oxide. In a further variation, the extended releaseformulation comprises sialic acid, or a pharmaceutically acceptable saltthereof, carrageenan, sodium alginate, either hypromellose orpolyethylene oxide, magnesium strearate and microcrystalline colluloseand colloiddal silicon dioxide. In one aspect, the extended releaseformulation is a formulation of Table E. In yet another aspect, theextended release formulation is a formulation of Table 8.

Particular extended release formulations include those listed in TableC, where the compositions comprise a therapeutic agent, a polymer 1, apolymer 2, either a polymer 3A or a polymer 3B, an excipient and alubricant, and it is understood that each and every combination of suchcomponents is intended the same as if each and every combination werespecifically and individually listed.

TABLE C Exemplary Extended Release Formulation Compositions. FormulationComponent Exemplary Specific Components Therapeutic mannosamine,N-acetyl mannosamine (ManNAc), ManNac-6-phosphate Agent (ManNAc-6-P),UDP-GlcNAc, N-acetylneuraminic acid (NeuAc), NeuAc- (A compound in9-phosphate (NeuAc-9-P), sialic acid (i.e., 5-N-acetylneuraminic acid),the sialic acid CMP-sialic acid, and/or derivatives thereof orpharmaceutically acceptable biosynthetic salts of the foregoing. pathwayor derivative thereof or salt of any of the foregoing) Polymer 1Carrageenan (e.g., iota, kappa or lambda carrageenan, or a salt thereof,such (Hydrocolloid as Viscarin GP-209, FMC BioPolymer) Polymer) Polymer2 Alginate or salt thereof (e.g., sodium, potassium or ammonium alginatesalt (Anionic, pH- such as Protanal ®, FMC BioPolymer), carboxymethylcellulose or salt dependent, gel thereof (e.g., sodium carboxymethylcellulose). forming co- polymer) Polymer 3A Hypromellose (e.g.,hypromellose Type 2208, E and K series such as for (Water-swellable,example, Dow Chemical Company's (Midland, Mich. USA) or Aqualon's pHindependent (with a North American presence in Wilmington, Del.) E4M,E10M, polymer) K100LV, K4M, K15M, K25M, K100M, K200M and mixtures ofvarious molecular weights and grades); hydroxypropyl ethyl cellulose(Ethocel polymers 7FP, 10FP and 100FP and Aqualon's polymers T10EC, N7,N10, N17, N22, N50, N100 and N200, and mixtures thereof), hydroxypropylcellulose (Aqualon's HPC polymers MF and MXF (mol. wt. 580,000) and KFand HXF (mol. wt. 1,150,000), and mixtures thereof); hydroxyethylcellulose (e.g., Aqualon's Natrasol polymers HHX (mol. Wt. 1,300,000),HX (mol. wt. 1,000,000), H (mol. wt. 1,000,000), M (mol. wt. 720,000 andG (mol. wt. 1,150,000), and mixtures thereof); methyl cellulose..Polymer 3B Polyhydroxyethyle methylacrylate (PHEMA), polyvinyl alcohol(PVA), (Hydrogel) polyvinyl pyrrolidone (PVP), polyethylene oxide (PEO),polyacrylamide (PA), polyethylene oxide (e.g., Polyox ™ water solubleresin, Dow Chemical Company, Mich., USA). Excipient lactose,microcrystalline cellulose, corn starch, potato starch, wheat starch,sucrose, D-mannitol, precipitated calcium carbonate, dextrin,pre-gelatinized starch, microcrystalline cellulose and colloidal silicondioxide (e.g., ProSolv ® SMCC HD90) and combinations thereof. LubricantStearate salt (such as magnesium stearate (e.g., HyQual ®) and calciumstearate), talc, light anhydrous silicic acid, and solid polyethylglycols and combinations thereof.

In one variation, an extended release formulation is a composition asdetailed in Table C, wherein the composition comprises the formulationcomponents in any one of the weight percent ranges depicted in Table D.It is understood that each and every combination of such components andweight percentages is intended the same as if each and every combinationof component and weight percentage were specifically and individuallylisted.

TABLE D Exemplary Weight Percent of Certain Components for Use inExtended Release Formulations Formulation Component Exemplary SpecificComponents Exemplary w/w % Therapeutic mannosamine, N-acetyl mannosamineFrom about 20 to about 80; from Agent (ManNAc), ManNac-6-phosphate about20 to about 60; from about (A compound (ManNAc-6-P), UDP-GlcNAc, N- 20to about 50; from about 20 to in the sialic acetylneuraminic acid(NeuAc), NeuAc-9- about 40; from about 20 to about acid phosphate(NeuAc-9-P), sialic acid (i.e., 5- 30; from about 15 to about 60;biosynthetic N-acetylneuraminic acid), CMP-sialic acid, from about 15 toabout 50; from pathway or and/or derivatives thereof or about 15 toabout 40; from about derivative pharmaceutically acceptable salts of the25 to about 60; from about 25 to thereof or salt foregoing. about 50;from 25 to about 40; of any of the from about 25 to about 30; fromforegoing) about 30 to about 60; from about 30 to about 50; from about30 to about 45; from about 30 to about 40; from about 35 to about 60;from about 35 to about 50; from about 35 to about 45; from about 40 toabout 45; about any of 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49 and 50. Polymer 1 Carrageenan (e.g., iota, kappa or lambdaFrom about 1 to about 10; from (Hydrocolloid carrageenan, or a saltthereof, such as about 1 to about 5; from about 3 Polymer) ViscarinGP-209, FMC BioPolymer) to about 8; from about 4 to about 6; about anyof 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. Polymer 2 Alginate or salt thereof(e.g., sodium, From about 15 to about 30; from (Anionic, pH- potassiumor ammonium alginate salt such about 15 to about 25; from aboutdependent, gel as Protanal ®, FMC BioPolymer), 15 to about 20; fromabout 20 to forming co- carboxymethyl cellulose or salt thereof about30; from about 20 to about polymer) (e.g., sodium carboxymethylcellulose). 25; from about 2o to about 23; about any of 15, 16, 17, 18,19, 20, 21, 22, 23, 24 and 25. Polymer 3A Hypromellose (e.g.,hypromellose Type From about 20 to about 50; from (Water- 2208, E and Kseries such as for example, about 20 to about 40, from about swellable,pH Dow Chemical Company's (Midland, 20 to about 30; from about 20 toindependent Mich. USA) or Aqualon's (with a North about 25; from about25 to about polymer) American presence in Wilmington, Del.) 30; fromabout 22 to about 27; E4M, E10M, K100LV, K4M, K15M, about any of 20, 21,22, 23, 24, K25M, K100M, K200M and mixtures of 25, 26, 27, 28, 29 and30. various molecular weights and grades); hydroxypropyl ethyl cellulose(Ethocel polymers 7FP, 10FP and 100FP and Aqualon's polymers T10EC, N7,N10, N17, N22, N50, N100 and N200, and mixtures thereof), hydroxypropylcellulose (Aqualon's HPC polymers MF and MXF (mol. wt. 580,000) and KFand HXF (mol. wt. 1,150,000), and mixtures thereof); hydroxyethylcellulose (e.g., Aqualon's Natrasol polymers HHX (mol. Wt. 1,300,000),HX (mol. wt. 1,000,000), H (mol. wt. 1,000,000), M (mol. wt. 720,000 andG (mol. wt. 1,150,000), and mixtures thereof); methyl cellulose..Polymer 3B Polyhydroxyethyle methylacrylate From about 20 to about 30;from (Hydrogel) (PHEMA), polyvinyl alcohol (PVA), about 20 to about 25;from about polyvinyl pyrrolidone (PVP), polyethylene 25 to about 30;from about 22 to oxide (PEO), polyacrylamide (PA), about 27; about anyof 20, 21, polyethylene oxide (e.g., Polyox ™ water 22, 23, 24, 25, 26,27, 28, 29 and soluble resin, Dow Chemical Company, 30. Mich., USA).Excipient lactose, microcrystalline cellulose, corn From about 1 toabout 20 or starch, potato starch, wheat starch, sucrose, about 1 toabout 15 or about 1 to D-mannitol, precipitated calcium about 10 orabout 1 to about 5 or carbonate, dextrin, pre-gelatinized starch, about5 to about 20 or about 5 to microcrystalline cellulose and colloidalabout 15 or about 5 to about 10 silicon dioxide (e.g., ProSolv ® SMCC orabout any one of 1, 2, 3, 4, 5, HD90) and combinations thereof. 6, 7, 8,9 or 10 weight percent. Lubricant Stearate salt (such as magnesiumstearate From about 0.1 to about 2 or (e.g., HyQual ®) and calciumstearate), talc, about 0.1 to about 1.5 or about light anhydrous silicicacid, and solid 0.1 to about 1.0 or about .01 to polyethyl glycols andcombinations about .09-5 or about 0.1 to about thereof. 0.8 or about 0.1to about 0.7 or about 0.1 to about 0.6 or about 0.1 to about 0.5 orabout 0.2 to about 0.8 or about 0.3 to about 0.7 or about 0.4 to about0.6 or about any one of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or1.0 weight percent.

In another variation, extended release formulations are provided whereinthe formulation comprises a therapeutic agent of Table C, a polymer 1 ofTable C, a polymer 2 of Table C, a Polymer 3A or a polymer 3B of TableC, an excipient of Table C and a lubricant of Table C, wherein thecomponents are present in the composition in the following weightpercent ratios. In one aspect, the weight percent ratio ofLubricant:Polymer 1:Excipient:Polymer 2:Polymer 3A or 3B:TherapeuticAgent is about 1:8:10:40:50:85 or about 1:8.5:10.5:42.5:51:86.5 or about1:8.4:10.6:42.4:51:86.6.

In any of the formulae detailed herein, including but not limited to theformulations in any of Tables A-D, in one aspect the therapeutic agentis sialic acid or ManNAc or a pharmaceutically acceptable salt thereofor a combination of sialic acid or ManNAc. In a particular aspect of anyof the formulations detailed herein, including but not limited to theformulations in any of Tables A-D, the therapeutic agent is sialic acid,or a pharmaceutically acceptable salt thereof.

Particular extended release formulations of sialic acid are provided inTable E. In one variation, ManNAc may be used in place of sialic acid inthe formulations of Table E.

TABLE E Exemplary Extended Release Formulations of sialic acid.Formulation Component Exemplary w/w % Sialic acid, or From about 20 toabout 80; from about 20 to about 60; from about 20 pharmaceutically toabout 50; from about 20 to about 40; from about 20 to about 30;acceptable salt thereof from about 15 to about 60; from about 15 toabout 50; from about 15 to about 40; from about 25 to about 60; fromabout 25 to about 50; from 25 to about 40; from about 25 to about 30;from about 30 to about 60; from about 30 to about 50; from about 30 toabout 45; from about 30 to about 40; from about 35 to about 60; fromabout 35 to about 50; from about 35 to about 45; from about 40 to about45; about any of 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49 and 50. Carrageenan (e.g., From about 1 to about 10; from about 1 toabout 5; from about 3 to lambda carrageenan about 8; from about 4 toabout 6; about any of 1, 2, 3, 4, 5, 6, 7, 8, 9 such as Viscarin GP- and10. 209, FMC BioPolymer) Alginate or a salt From about 15 to about 30;from about 15 to about 25; from about 15 thereof (e.g., sodium, to about20; from about 20 to about 30; from about 20 to about 25; potassium orfrom about 2o to about 23; about any of 15, 16, 17, 18, 19, 20, 21, 22,ammonium alginate 23, 24 and 25. salt such as Protanal ® LF 120M)Hypromellose (such as From about 20 to about 50; from about 20 to about40, from about 20 hypromellose Type to about 30; from about 20 to about25; from about 25 to about 30; 2208, e.g., from about 22 to about 27;about any of 20, 21, 22, 23, 24, 25, 26, 27, Methocel ®K100 M 28, 29 and30. Premium CR) Polyethylene oxide From about 20 to about 30; from about20 to about 25; from about 25 (such as Polyethylene to about 30; fromabout 22 to about 27; about any of 20, 21, 22, 23, 24, Oxide WSR, e.g.,25, 26, 27, 28, 29 and 30. Polyox ™ water soluble resin, Dow ChemicalCompany, Mich., USA) Microcrystalline From about 1 to about 20 or about1 to about 15 or about 1 to about 10 cellulose and colloidal or about 1to about 5 or about 5 to about 20 or about 5 to about 15 or silicondioxide (e.g., about 5 to about 10 or about any one of 1, 2, 3, 4, 5, 6,7, 8, 9 or 10 ProSolv ® SMCC weight percent. HD90) Stearate salt (suchas From about 0.1 to about 2 or about 0.1 to about 1.5 or about 0.1 tomagnesium stearate about 1.0 or about .01 to about .09-5 or about 0.1 toabout 0.8 or about (e.g., HyQual ®) 0.1 to about 0.7 or about 0.1 toabout 0.6 or about 0.1 to about 0.5 or about 0.2 to about 0.8 or about0.3 to about 0.7 or about 0.4 to about 0.6 or about any one of 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0 weight percent.

The components used to formulate the extended release pharmaceuticalformulations are preferably of high purity and are substantially free ofpotentially harmful contaminants (e.g., at least National Food (NF)grade, generally at least analytical grade, and more typically at leastpharmaceutical grade). Moreover, pharmaceutical formulations intendedfor in vivo use are usually sterile. To the extent that a given compoundmust be synthesized prior to use, the resulting product is typicallysubstantially free of any potentially toxic agents which may be presentduring the synthesis or purification process. Compositions for parentaladministration are also sterile, substantially isotonic and made underGMP conditions.

The blends of the extended release formulation may have a particle sizewith a majority of particles being retained by a sieve size of 45 Insome embodiments, the blends of the extended release formulation have aparticle size with at least any one of 10%, 30%, 40%, 50% of particlesretained by a sieve size of 45 μm.

The extended release pharmaceutical formulations as described herein maybe formulated into preparations in solid, semi-solid, liquid or gaseousforms, such as tablets, capsules, powders, granules, ointments,solutions, suppositories, injections, inhalants, gels, microspheres, andaerosols. In some embodiments of any of the extended releasepharmaceutical formulations described herein, the extended releasepharmaceutical formulations is formulated for administration by avariety of routes including oral, parenteral (including subcutaneous,intravenous, intramuscular and intraperitoneal), rectal, dermal,transdermal, intrathoracic, intrapulmonary and intranasal (respiratory)routes. In some embodiments, the extended release pharmaceuticalformulation is formulated for oral administration.

Any of the extended release formulations detailed herein may in onevariation be formulated for oral administration. For example, any of theformulations provided under the heading “Extended Release Formulations,”including but not limited to any of the formulations set forth in TablesA-E may in one variation be a formulation that is suitable for oraladministration. A formulation that is suitable for oral administrationmay be formulated as a solid oral dosage form, such as a tablet or acapsule comprising the formulation as a powder. In one aspect, a solidoral dosage form of an extended release formulation is provided whereinthe solid oral dosage form comprises any formulation provided herein(including but not limited to the formulations set forth in any one ofTables A-E) in tablet form, wherein the tablet further comprises acoating (e.g., Opadry-II White). In one varation, the extended releaseformulation comprises sialic acid, or a pharmaceutically acceptable saltthereof, as the therapeutic agent. In another variation, the extendedrelease formulation comprises MaNAc, or a pharmaceutically acceptablesalt thereof, as the therapeutic agent. In another variation, theextended release formulation comprises sialic acid, or apharmaceutically acceptable salt thereof and MaNAc, or apharmaceutically acceptable salt thereof, as the therapeutic agents. Inanother variation, the extended release formulation comprises a prodrugof one or more compounds in the sialic acid biosynthetic pathway such asa prodrug of sialic acid, or a pharmaceutically acceptable salt thereof,as the therapeutic agent. In another variation, the extended releaseformulation comrpises sialic acid, or a pharmaceutically acceptable saltthereof, as the therapeutic agent and further comprises a hydocolloidpolymer, an anionic, pH-dependent gel forming co-polymer and a waterswellable, pH independent polymer and optionally further comprises alubricant and/or an excipient. In a particular variation, the extendedrelease formulation comprises sialic acid, or a pharmaceuticallyacceptable salt thereof, carrageenan, sodium alginate and eitherhypromellose or polyethylene oxide. In a further variation, the extendedrelease formulation comprises sialic acid, or a pharmaceuticallyacceptable salt thereof, carrageenan, sodium alginate, eitherhypromellose or polyethylene oxide, magnesium strearate andmicrocrystalline collulose and colloiddal silicon dioxide. In oneaspect, the extended release formulation is a formulation of Table E. Inyet another aspect, the extended release formulation is a formulation ofTable 8.

For oral administration, the sialic acid biosynthetic pathway orderivative thereof as described herein can be administered in soliddosage forms, such as capsules, tablets, and powders, or in liquiddosage forms, such as elixirs, syrups, and suspensions.

In some embodiments, the pharmaceutical formulations comprise anenteric-coating. Numerous types of acid-resistant enteric coatings areavailable. Examples of the acid-resistant coatings include celluloseacetate phthalate, polyvinyl acetate phthalate, shellac, an acrylic acidhomopolymer or copolymer, a methacrylic acid homopolymer or copolymer,cellulose acetate trimellitate, hydroxypropyl methylcellulose phthalateor a combination of thereof. A number of copolymers of methacrylic acidare known in the art and are commercially available. Examples of suchpolymers are copolymers of methylmethacrylate and methacrylic acid andcopolymers of ethylacrylate and methacrylic acid, and sold under thetradename Eudragit (Rohm GmbH & Co. KG): examples include Eudragit® L100-55, Eudragit® L 30D-55, Eudragit® L 100, Eudragit® S 100-55 andEudragit® FS 30D. In some embodiments, the enteric coating comprises oneor more of titanium dioxide, polydextrose, hypromellose, triacetin andmacrogol/PEG. In some embodiments, the enteric coating is Opadry® IIWhite. In some embodiments, the enteric coating (e.g., Opadry® II White)comprises about 1-5% w/w of the extended release formulation. In someembodiments, the enteric coating (e.g., Opadry® II White) comprisesabout 1-5% w/w of the extended release formulation.

An enteric coating can also be a time-release coating. The time-releasecoatings are degraded away at a relatively constant rate until thecoatings dissolve sufficiently for the time-release coatings to rupture.Thus, the time required for the rupture of the enteric coatings islargely time-dependent (i.e., thickness), and largely pH independent.Examples of time-release coating materials include cellulose acetate,cellulose acetate butyrate, cellulose acetate propionate, EC, andcopolymers of acrylate and methacrylates with quaternary ammonium groupssuch as Eudragit® RL and Eudragit® RS and Eudragit® NE30-D.

The extended release pharmaceutical formulations can be furthersubjected to a process of film coating. For the film coating agent, anenteric or non-enteric film coating agent may be used, and the entericfilm coating agent can be cellulose acetate phthalate (CAP), polyvinylacetate phthalate (PVAP), a methacrylate polymer (Eudragit L, S), or thelike, while the non-enteric film coating agent can behydroxypropylcellulose (HPC), MC, EC, HPMC, povidone, PVA, CA, shellac,or the like. The process of coating can be performed by, for example, apan coating method, a fluidized bed coating method, a compressioncoating method, or the like.

Coated tablets of the extended release formulation may be prepared invarious sizes. For example, the coated tablets may have a length ofabout 16-20 mm, a width of about 7-12 mm and a thickness of about 5-8mm. In some embodiments, the coated tablets have a length of about 17.7mm, a width of about 9.1 mm and a thickness of about 6.7 mm. In someembodiments, the coated tablets have a length of about 19.3 mm, a widthof about 9.7 mm and a thickness of about 8.0 mm.

In embodiments of any of the methods, the extended release formulationcomprises a drug load of about 30-60% (e.g., sialic acid and/or ManNAc),about 20-30% w/w of a water-swellable, pH independent polymer (e.g.hypromellose), about 20-25% w/w of an anionic, pH-dependent, gel-formingcopolymer (e.g., an alginate salt), about 1-5% w/w of a hydrocolloidpolymer (e.g., a carrageenan), about 1-10% w/w of microcrystallinecellulose and colloidal silicon dioxide (e.g., Prosolv® SMCC HD90),about 0.1-1% w/w/magnesium stearate (e.g. HyQual®), and about 1-5% of anenteric coating (e.g. Opadry® II White). In some embodiments, theextended release formulation comprises a drug load of about 30-60%(e.g., sialic acid and/or ManNAc), about 20-30% w/w hypromellose (e.g.hypromellose Type 2208 or Methocel K100M), about 20-25% w/w sodiumalginate (e.g. Protanal), about 1-5% w/w lamda carrageenan (e.g.Viscarin GP-209), about 1-10% w/w of microcrystalline cellulose andcolloidal silicon dioxide (e.g., Prosolv® SMCC HD90), about 0.1-1%w/w/magnesium stearate (e.g. HyQual®), and about 1-5% of an entericcoating (e.g. Opadry® II White). In some embodiments, the extendedrelease formulation comprises a drug load of about 30-60% (e.g., sialicacid and/or ManNAc), about 25% w/w of a water-swellable, pH independentpolymer (e.g. hypromellose), about 21% w/w of an anionic, pH-dependent,gel-forming copolymer (e.g., an alginate salt), about 4% w/w of ahydrocolloid polymer (e.g., a carrageenan), about 5% w/w ofmicrocrystalline cellulose and colloidal silicon dioxide (e.g., Prosolv®SMCC HD90), about 0.5% w/w/magnesium stearate (e.g. HyQual®), and about3.5% of an enteric coating (e.g. Opadry® II White). In some embodiments,the extended release formulation comprises a drug load of about 30-60%(e.g., sialic acid and/or ManNAc), about 25% w/w hypromellose (e.g.hypromellose Type 2208 or Methocel K100M), about 21% w/w sodium alginate(e.g. Protanal), about 4% w/w lamda carrageenan (e.g. Viscarin GP-209),about 5% w/w of microcrystalline cellulose and colloidal silicon dioxide(e.g., Prosolv® SMCC HD90), about 0.5% w/w/magnesium stearate (e.g.HyQual®), and about 3.5% of an enteric coating (e.g. Opadry® II White).

In embodiments of any of the methods, the extended release formulationcomprises a drug load of about 30-60% (e.g., sialic acid and/or ManNAc),about 20-30% w/w of a hydrogel (e.g. polyethylene oxide), about 20-25%w/w of an anionic, pH-dependent, gel-forming copolymer (e.g., analginate salt), about 1-5% w/w of a hydrocolloid polymer (e.g., acarrageenan), about 1-10% w/w of microcrystalline cellulose andcolloidal silicon dioxide (e.g., Prosolv® SMCC HD90), about 0.1-1%w/w/magnesium stearate (e.g. HyQual®), and about 1-5% of an entericcoating (e.g. Opadry® II White). In some embodiments, the extendedrelease formulation comprises a drug load of about 30-60% (e.g., sialicacid and/or ManNAc), about 20-30% w/w polyethylene oxide (e.g. PolyoxWSR), about 20-25% w/w sodium alginate (e.g. Protanal), about 1-5% w/wlamda carrageenan (e.g. Viscarin GP-209), about 1-10% w/w ofmicrocrystalline cellulose and colloidal silicon dioxide (e.g., Prosolv®SMCC HD90), about 0.1-1% w/w/magnesium stearate (e.g. HyQual®), andabout 1-5% of an enteric coating (e.g. Opadry® II White). In someembodiments, the extended release formulation comprises a drug load ofabout 30-60% (e.g., sialic acid and/or ManNAc), about 25% w/w of ahydrogel (e.g. polyethylene oxide), about 21% w/w of an anionic,pH-dependent, gel-forming copolymer (e.g., an alginate salt), about 4%w/w of a hydrocolloid polymer (e.g., a carrageenan), about 5% w/w ofmicrocrystalline cellulose and colloidal silicon dioxide (e.g., Prosolv®SMCC HD90), about 0.5% w/w/magnesium stearate (e.g. HyQual®), and about3.5% of an enteric coating (e.g. Opadry® II White). In some embodiments,the extended release formulation comprises a drug load of about 30-60%(e.g., sialic acid and/or ManNAc), about 25% w/w polyethylene oxide(e.g. Polyos WSR), about 21% w/w sodium alginate (e.g. Protanal), about4% w/w lamda carrageenan (e.g. Viscarin GP-209), about 5% w/w ofmicrocrystalline cellulose and colloidal silicon dioxide (e.g., Prosolv®SMCC HD90), about 0.5% w/w/magnesium stearate (e.g. HyQual®), and about3.5% of an enteric coating (e.g. Opadry® II White).

Extended Release Formulation Characteristics

Any of the extended release formulations detailed herein, including butnot limited to those detailed under the heading “Extended RelaseFormulation” and “Additional Formulation Components” (e.g., any of theformulations of Tables A-E) may exhibit any of the characteristicsdetailed herein and below. In a particular variation, any of theextended release formulations detailed herein may exhibit any one ormore of the following characteristics: (i) capable of delivering to anindividual in need thereof a therapeutically effective amount of one ormore compounds in the sialic acid pathway or derivatives thereof over aperiod of greater than about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours; (ii)capable of delivering to an individual in need thereof a substantiallyconstant therapeutically effective amount of one or more compounds inthe sialic acid pathway or derivatives thereof over a period of greaterthan about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, or 24 hours; (iii) capable of delivering toan individual in need thereof a therapeutically effective amount of oneor more compounds in the sialic acid pathway or derivatives thereof witha T_(max) of between about any of 2-4 hours, 3-4 hours, 6-8 hours, 6-12hours, 6-15 hours, 12-18 hours, or 18-24 hours; (iv) capable ofdelivering to an individual in need thereof a therapeutically effectiveamount of one or more compounds in the sialic acid pathway orderivatives thereof with a C_(max) of about 0.5-100 μg/mL; (v) capableof delivering to an individual in need thereof a therapeuticallyeffective amount of one or more compounds in the sialic acid pathway orderivatives thereof with a trough level of about 0.1-20 μg/mL; (vi)capable of delivering to an individual in need thereof a therapeuticallyeffective amount of one or more compounds in the sialic acid pathway orderivatives thereof with less than about any of 10%, 20%, 30%, 40%, 50%,60%, or 70% excreted after one hour; (vii) capable of delivering to anindividual in need thereof between about any of 0.01-750 mg/kg/day,0.5-500 mg/kg/day, 1-250 mg/kg/day, 2.5-100 mg/kg/day, or 5-50 mg/kg/dayof one or more compounds in the sialic acid pathway or derivativesthereof or a pharmaceutically acceptable salt of the foregoing; (viii)capable of delivering to an individual in need thereof between about anyof 0.01-750 mg/kg/day, 0.5-500 mg/kg/day, 1-250 mg/kg/day, 2.5-100mg/kg/day, or 5-50 mg/kg/day of one or more compounds in the sialic acidpathway or derivatives thereof or a pharmaceutically acceptable salt ofthe foregoing; (ix) has an absolute bioavailability of about 1 to about50%; (x) has a bioavailability based on sialic acid levels in the urineof about 0.5 to about 100%; (xi) has a mean residence time (MRT) of atleast about 3.5 hours. In one varation, the extended release formulationcomprises sialic acid, or a pharmaceutically acceptable salt thereof, asthe therapeutic agent. In another variation, the extended releaseformulation comprises MaNAc, or a pharmaceutically acceptable saltthereof, as the therapeutic agent. In another variation, the extendedrelease formulation comprises sialic acid, or a pharmaceuticallyacceptable salt thereof and MaNAc, or a pharmaceutically acceptable saltthereof, as the therapeutic agents. In another variation, the extendedrelease formulation comprises a prodrug of one or more compounds in thesialic acid biosynthetic pathway such as a prodrug of sialic acid, or apharmaceutically acceptable salt thereof, as the therapeutic agent. Inanother variation, the extended release formulation comrpises sialicacid, or a pharmaceutically acceptable salt thereof, as the therapeuticagent and further comprises a hydocolloid polymer, an anionic,pH-dependent gel forming co-polymer and a water swellable, pHindependent polymer and optionally further comprises a lubricant and/oran excipient. In a particular variation, the extended releaseformulation comprises sialic acid, or a pharmaceutically acceptable saltthereof, carrageenan, sodium alginate and either hypromellose orpolyethylene oxide. In a further variation, the extended releaseformulation comprises sialic acid, or a pharmaceutically acceptable saltthereof, carrageenan, sodium alginate, either hypromellose orpolyethylene oxide, magnesium strearate and microcrystalline colluloseand colloiddal silicon dioxide. In one aspect, the extended releaseformulation is a formulation of Table E. In yet another aspect, theextended release formulation is a formulation of Table 8.

In embodiments of any of the extended release formulations, the extendedrelease formulation is capable of delivering to an individual in needthereof a therapeutically effective amount of one or more compounds inthe sialic acid pathway or derivatives thereof over a period of greaterthan about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, or 24 hours. In some embodiments, theextended release formulation is capable of delivering to an individualin need thereof a therapeutically effective amount of one or morecompounds in the sialic acid pathway or derivatives thereof over aperiod of greater than about 12 hours or greater than about 24 hours. Inembodiments of any of the extended release formulations, the extendedrelease formulation is capable of delivering to an individual in needthereof a therapeutically effective amount of one or more compounds inthe sialic acid pathway or derivatives thereof over a period of betweenabout any of 1-24 hours, 4-24 hours, 6-24 hours, 8-24 hours, or 12-24hours. In embodiments of any of the extended release formulations, theextended release formulation is capable of delivering to an individualin need thereof a therapeutically effective amount of one or morecompounds in the sialic acid pathway or derivatives thereof over aperiod of about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours. In some embodiments,the extended release formulation is capable of delivering to anindividual in need thereof a therapeutically effective amount of one ormore compounds in the sialic acid pathway or derivatives thereof over aperiod of about 12 hours or about 24 hours. In some embodiments, thetherapeutically effective amount is delivered to the bloodstream of theindividual. In some embodiments, the therapeutically effective amount isdelivered to muscle tissue of the individual. In some embodiments, theone or more compounds in the sialic acid pathway or derivatives thereofinclude ManNAc or a derivative thereof and/or sialic acid or aderivative thereof. For example, in some embodiments, the extendedrelease formulation is capable of delivering to an individual in needthereof a therapeutically effective amount of ManNAc and/or sialic acidto muscle tissue of the individual over a period of between about any of1-24 hours, 4-24 hours, 6-24 hours, 8-24 hours, or 12-24 hours. In onevaration, the extended release formulation comprises sialic acid, or apharmaceutically acceptable salt thereof, as the therapeutic agent. Inanother variation, the extended release formulation comprises MaNAc, ora pharmaceutically acceptable salt thereof, as the therapeutic agent. Inanother variation, the extended release formulation comprises sialicacid, or a pharmaceutically acceptable salt thereof and MaNAc, or apharmaceutically acceptable salt thereof, as the therapeutic agents.

In embodiments of any of the extended release formulations, the extendedrelease formulation is capable of delivering to an individual in needthereof a substantially constant (i.e., without large burst of drugavailability and deficiencies in drug availability to the blood and/ortissues of interest (e.g., muscle tissue)) therapeutically effectiveamount of one or more compounds in the sialic acid pathway orderivatives thereof over a period of greater than about any of 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, or 24 hours. In embodiments of any of the extended releaseformulations, the extended release formulation is capable of deliveringto an individual in need thereof a substantially constanttherapeutically effective amount of one or more compounds in the sialicacid pathway or derivatives thereof over a period of between about anyof 1-24 hours, 4-24 hours, 6-24 hours, 8-24 hours, or 12-24 hours. Inembodiments of any of the extended release formulations, the extendedrelease formulation is capable of delivering to an individual in needthereof a substantially constant therapeutically effective amount of oneor more compounds in the sialic acid pathway or derivatives thereof overa period of about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours. In some embodiments,the one or more compounds in the sialic acid pathway or derivativesthereof include ManNAc or a derivative thereof and/or sialic acid or aderivative thereof. For example, in some embodiments, the extendedrelease formulation is capable of delivering to an individual in needthereof a substantially constant therapeutically effective amount ofManNAc and/or sialic acid to muscle tissue of the individual over aperiod of between about any of 1-24 hours, 4-24 hours, 6-24 hours, 8-24hours, or 12-24 hours. In one varation, the extended release formulationcomprises sialic acid, or a pharmaceutically acceptable salt thereof, asthe therapeutic agent. In another variation, the extended releaseformulation comprises MaNAc, or a pharmaceutically acceptable saltthereof, as the therapeutic agent. In another variation, the extendedrelease formulation comprises sialic acid, or a pharmaceuticallyacceptable salt thereof and MaNAc, or a pharmaceutically acceptable saltthereof, as the therapeutic agents.

In embodiments of any of the extended release formulations, the extendedrelease formulation is capable of delivering to an individual in needthereof a therapeutically effective amount of one or more compounds inthe sialic acid pathway or derivatives thereof with a T_(max) of betweenabout any of 2-4 hours, 3-4 hours, 6-8 hours, 6-12 hours, 6-15 hours,12-18 hours, or 18-24 hours. In some embodiments, the extended releaseformulation is capable of delivering to an individual in need thereof atherapeutically effective amount of one or more compounds in the sialicacid pathway or derivatives thereof with a T_(max) of about 2-4 hours.In some embodiments, the extended release formulation is capable ofdelivering to an individual in need thereof a therapeutically effectiveamount of one or more compounds in the sialic acid pathway orderivatives thereof with a T_(max) of about 4-6 hours. In someembodiments, the extended release formulation is capable of deliveringto an individual in need thereof a therapeutically effective amount ofone or more compounds in the sialic acid pathway or derivatives thereofwith a T_(max) of between about 2 and about 8 hours. In someembodiments, the one or more compounds in the sialic acid pathway orderivatives thereof include ManNAc or a derivative thereof and/or sialicacid or a derivative thereof. In some embodiments, the one or morecompounds in the sialic acid pathway or derivatives thereof includeManNAc and/or sialic acid. In some embodiments, the one or morecompounds in the sialic acid pathway is sialic acid. In one varation,the extended release formulation comprises sialic acid, or apharmaceutically acceptable salt thereof, as the therapeutic agent. Inanother variation, the extended release formulation comprises MaNAc, ora pharmaceutically acceptable salt thereof, as the therapeutic agent. Inanother variation, the extended release formulation comprises sialicacid, or a pharmaceutically acceptable salt thereof and MaNAc, or apharmaceutically acceptable salt thereof, as the therapeutic agents.

In embodiments of any of the extended release formulations, the extendedrelease formulation is capable of delivering to an individual in needthereof a therapeutically effective amount of one or more compounds inthe sialic acid pathway or derivatives thereof with a C_(max) of about0.5-100 μg/mL. In some embodiments, the extended release formulation iscapable of delivering to an individual in need thereof a therapeuticallyeffective amount of one or more compounds in the sialic acid pathway orderivatives thereof with a C_(max) of about any one 0.5-80 μg/mL, 0.5-60μg/mL, 0.5-40 μg/mL or 0.5-20 μg/mL. In some embodiments, the extendedrelease formulation is capable of delivering to an individual in needthereof a therapeutically effective amount of one or more compounds inthe sialic acid pathway or derivatives thereof with a C_(max) of about0.5-40 μg/mL. In some embodiments, the extended release formulation iscapable of delivering to an individual in need thereof a therapeuticallyeffective amount of one or more compounds in the sialic acid pathway orderivatives thereof with a C_(max) of about any one of 0.5-35 μg/mL,0.5-30 μg/mL, 0.5-25 μg/mL, 1-40 μg/mL, 2.5-40 μg/mL, 5-40 μg/mL, 0.5-35μg/mL, 1-35 μg/mL, 2.5-35 μg/mL, 5-35 μg/mL, 0.5-30 μg/mL, 1-30 μg/mL,2.5-30 μg/mL, 5-30 μg/mL, 0.5-25 μg/mL, 1-25 μg/mL, 2.5-25 μg/mL, or5-25 μg/mL. In some embodiments, the extended release formulation iscapable of delivering to an individual in need thereof a therapeuticallyeffective amount of one or more compounds in the sialic acid pathway orderivatives thereof with a C_(max) of about 0.5-20 μg/mL. In someembodiments, the extended release formulation is capable of deliveringto an individual in need thereof a therapeutically effective amount ofone or more compounds in the sialic acid pathway or derivatives thereofwith a C_(max) of about any one of 0.5-15 μg/mL, 0.5-10 μg/mL, 1-20μg/mL, 2.5-20 μg/mL, 5-20 μg/mL, 0.5-15 μg/mL, 1-15 μg/mL, 2.5-15 μg/mL,5-15 μg/mL, 0.5-10 μg/mL, 1-10 μg/mL, 2.5-10 μg/mL, or 5-10 μg/mL. Insome embodiments, the one or more compounds in the sialic acid pathwayor derivatives thereof include ManNAc and/or sialic acid. In someembodiments, the one or more compounds in the sialic acid pathway orderivatives thereof include sialic acid. In one varation, the extendedrelease formulation comprises sialic acid, or a pharmaceuticallyacceptable salt thereof, as the therapeutic agent. In another variation,the extended release formulation comprises MaNAc, or a pharmaceuticallyacceptable salt thereof, as the therapeutic agent. In another variation,the extended release formulation comprises sialic acid, or apharmaceutically acceptable salt thereof and MaNAc, or apharmaceutically acceptable salt thereof, as the therapeutic agents.

In embodiments of any of the extended release formulations, the extendedrelease formulation is capable of delivering to an individual in needthereof a therapeutically effective amount of one or more compounds inthe sialic acid pathway or derivatives thereof with a trough level ofabout 0.1-20 μg/mL. In embodiments of any of the extended releaseformulations, the extended release formulation is capable of deliveringto an individual in need thereof a therapeutically effective amount ofone or more compounds in the sialic acid pathway or derivatives thereofwith a trough level of about any one of 0.1-15 μg/mL, 0.1-10 μg/mL,0.1-5 μg/mL, 0.5-20, μg/mL, 0.5-15 μg/mL, 0.5-10 μg/mL, 0.5-5 μg/mL,1-20 μg/mL, 1-15 μg/mL, 1-10 μg/mL, or 1-5 μg/mL or about any one of0.1, 0.5, 1 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 7, 8, 9, 10, 11, 12,13, 14 or 15 μg/mL. In some embodiments, the one or more compounds inthe sialic acid pathway or derivatives thereof include ManNAc and/orsialic acid. In some embodiments, the one or more compounds in thesialic acid pathway or derivatives thereof include sialic acid. In onevaration, the extended release formulation comprises sialic acid, or apharmaceutically acceptable salt thereof, as the therapeutic agent. Inanother variation, the extended release formulation comprises MaNAc, ora pharmaceutically acceptable salt thereof, as the therapeutic agent. Inanother variation, the extended release formulation comprises sialicacid, or a pharmaceutically acceptable salt thereof and MaNAc, or apharmaceutically acceptable salt thereof, as the therapeutic agents.

In embodiments of any of the extended release formulations, the extendedrelease formulation is capable of delivering to an individual in needthereof a therapeutically effective amount of one or more compounds inthe sialic acid pathway or derivatives thereof with less than about anyof 10%, 20%, 30%, 40%, 50%, 60%, or 70% excreted after one hour. Inembodiments of any of the extended release formulations, the extendedrelease formulation is capable of delivering to an individual in needthereof a therapeutically effective amount of one or more compounds inthe sialic acid pathway or derivatives thereof with less than about anyof 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% excreted after fourhours. In embodiments of any of the extended release formulations, theextended release formulation is capable of delivering to an individualin need thereof a therapeutically effective amount of one or morecompounds in the sialic acid pathway or derivatives thereof with lessthan about any one of 2, 3, 4, or 5% excreted after 12 hours. In someembodiments, the one or more compounds in the sialic acid pathway orderivatives thereof include ManNAc or a derivative thereof and/or sialicacid or a derivative thereof. In some embodiments, the one or morecompounds in the sialic acid pathway or derivatives thereof includeManNAc and/or sialic acid. In some embodiments, the one or morecompounds in the sialic acid pathway or derivatives thereof includesialic acid. In one varation, the extended release formulation comprisessialic acid, or a pharmaceutically acceptable salt thereof, as thetherapeutic agent. In another variation, the extended releaseformulation comprises MaNAc, or a pharmaceutically acceptable saltthereof, as the therapeutic agent. In another variation, the extendedrelease formulation comprises sialic acid, or a pharmaceuticallyacceptable salt thereof and MaNAc, or a pharmaceutically acceptable saltthereof, as the therapeutic agents.

In embodiments of any of the extended release formulations, the extendedrelease formulation is capable of delivering to an individual in needthereof between about any of 0.1-50 g/day, 0.5-25 g/day, 1-15 g/day,1-10 g/day, or 2-5 g/day of one or more compounds in the sialic acidpathway or derivatives thereof. In some embodiments, the extendedrelease formulation is capable of delivering to an individual in needthereof between about 2 g/day and 5 g/day of one or more compounds inthe sialic acid pathway or derivatives thereof. In embodiments of any ofthe extended release formulations, the extended release formulation iscapable of delivering to an individual in need thereof between about anyof 0.01-750 mg/kg, 0.5-500 mg/kg, 1-250 mg/kg, 2.5-100 mg/kg, or 5-50mg/kg of one or more compounds in the sialic acid pathway or derivativesthereof. In some embodiments, the extended release formulation iscapable of delivering to an individual in need thereof between about 5mg/kg and 50 mg/kg of one or more compounds in the sialic acid pathwayor derivatives thereof. In some embodiments, the one or more compoundsin the sialic acid pathway or derivatives thereof include ManNAc or aderivative thereof and/or sialic acid or a derivative thereof. Forexample, in some embodiments, the extended release formulation iscapable of delivering to an individual in need thereof between about 5mg/kg and 50 mg/kg of ManNAc and/or sialic acid. In one varation, theextended release formulation comprises sialic acid, or apharmaceutically acceptable salt thereof, as the therapeutic agent. Inanother variation, the extended release formulation comprises MaNAc, ora pharmaceutically acceptable salt thereof, as the therapeutic agent. Inanother variation, the extended release formulation comprises sialicacid, or a pharmaceutically acceptable salt thereof and MaNAc, or apharmaceutically acceptable salt thereof, as the therapeutic agents.

In embodiments of any of the extended release formulations, the extendedrelease formulation is capable of delivering to an individual in needthereof between about any of 0.01-750 mg/kg/day, 0.5-500 mg/kg/day,1-250 mg/kg/day, 2.5-100 mg/kg/day, or 5-50 mg/kg/day of one or morecompounds in the sialic acid pathway or derivatives thereof. In someembodiments, the extended release formulation is capable of deliveringto an individual in need thereof between about 5 mg/kg/day and 50mg/kg/day of one or more compounds in the sialic acid pathway orderivatives thereof. In some embodiments, the one or more compounds inthe sialic acid pathway or derivatives thereof include ManNAc or aderivative thereof and/or sialic acid or a derivative thereof. Forexample, in some embodiments, the extended release formulation iscapable of delivering to an individual in need thereof between about 5mg/kg/day and 50 mg/kg/day of ManNAc and/or sialic acid. In onevaration, the extended release formulation comprises sialic acid, or apharmaceutically acceptable salt thereof, as the therapeutic agent. Inanother variation, the extended release formulation comprises MaNAc, ora pharmaceutically acceptable salt thereof, as the therapeutic agent. Inanother variation, the extended release formulation comprises sialicacid, or a pharmaceutically acceptable salt thereof and MaNAc, or apharmaceutically acceptable salt thereof, as the therapeutic agents.

In embodiments of any of the extended release formulations, the extendedrelease formulation has an absolute bioavailability of about 1-50%. Insome embodiments, the extended release formulation has an absolutebioavailability of about any one of 1-45%, 1-40%, 1-35%, 1-30%, 1-20%,1-10%. In some embodiments the extended release formulation has anabsolute bioavailability of about 1-25%. In some embodiments, theextended release formulation has an absolute bioavailability of aboutany one of 5, 10, 15, 20, 25 or 50%. In some embodiments, the one ormore compounds in the sialic acid pathway or derivatives thereof includeManNAc or a derivative thereof and/or sialic acid or a derivativethereof. In some embodiments, the one or more compounds in the sialicacid pathway or derivatives thereof include sialic acid or a derivativethereof. In one varation, the extended release formulation comprisessialic acid, or a pharmaceutically acceptable salt thereof, as thetherapeutic agent. In another variation, the extended releaseformulation comprises MaNAc, or a pharmaceutically acceptable saltthereof, as the therapeutic agent. In another variation, the extendedrelease formulation comprises sialic acid, or a pharmaceuticallyacceptable salt thereof and MaNAc, or a pharmaceutically acceptable saltthereof, as the therapeutic agents.

In embodiments of any of the extended release formulations, the extendedrelease formulation has a bioavailability based on sialic acid levels inthe urine of about 0.5-100%. In some embodiments, the extended releaseformulation has a bioavailability based on sialic acid levels in theurine of about any one of 0.5-2.5%, 1-2.5%, 2-8%, 2-12%, 2.5-20%,2.5-40%, 2.5-80%, 2.5-100%. In one varation, the extended releaseformulation comprises sialic acid, or a pharmaceutically acceptable saltthereof, as the therapeutic agent. In another variation, the extendedrelease formulation comprises MaNAc, or a pharmaceutically acceptablesalt thereof, as the therapeutic agent. In another variation, theextended release formulation comprises sialic acid, or apharmaceutically acceptable salt thereof and MaNAc, or apharmaceutically acceptable salt thereof, as the therapeutic agents.

In embodiments of any of the extended release formulations, the extendedrelease formulation has a mean residence time (MRT) of at least about3.5 hours. In some embodiments, the extended release formulation has aMRT of at least about any one of 3, 4, 4.5, 5, 5.5 or 6 hours. In someembodiments, the one or more compounds in the sialic acid pathway orderivatives thereof include ManNAc or a derivative thereof and/or sialicacid or a derivative thereof. In some embodiments, the one or morecompounds in the sialic acid pathway or derivatives thereof includesialic acid or a derivative thereof. In one varation, the extendedrelease formulation comprises sialic acid, or a pharmaceuticallyacceptable salt thereof, as the therapeutic agent. In another variation,the extended release formulation comprises MaNAc, or a pharmaceuticallyacceptable salt thereof, as the therapeutic agent. In another variation,the extended release formulation comprises sialic acid, or apharmaceutically acceptable salt thereof and MaNAc, or apharmaceutically acceptable salt thereof, as the therapeutic agents.

The extended release pharmaceutical formulation may be formulated forparenteral administration (e.g., by injection, for example, bolusinjection or continuous infusion) and may be presented in unit dose formin ampoules, pre-filled syringes, small volume infusion containers or inmulti-dose containers. The extended release pharmaceutical formulationmay be may form suspensions, solutions, or emulsions in oily or aqueousvehicles. Alternatively, the one or more compounds in the sialic acidbiosynthetic pathway or derivative thereof and other ingredients may bein powder form, obtained by aseptic isolation of sterile solid or bylyophilization from solution, for constitution with a suitable vehicle,e.g., sterile, pyrogen-free water, before use.

For topical administration, the extended release pharmaceuticalformulation may be formulated as is known in the art for directapplication to a target area. Forms chiefly conditioned for topicalapplication take the form, for example, of creams, milks, gels,dispersion or microemulsions, lotions thickened to a greater or lesserextent, impregnated pads, ointments or sticks, aerosol formulations(e.g., sprays or foams), soaps, detergents, lotions or cakes of soap.Other conventional forms for this purpose include wound dressings,coated bandages or other polymer coverings, ointments, creams, lotions,pastes, jellies, sprays, and aerosols. The extended releasepharmaceutical formulation may be delivered via patches or bandages fordermal administration. Ointments and creams may, for example, beformulated with an aqueous or oily base with the addition of suitablethickening and/or gelling agents. Drops, such as eye drops or nosedrops, may be formulated with the one or more compounds in the sialicacid biosynthetic pathway or derivative thereof in an aqueous ornon-aqueous base. Liquid sprays are conveniently delivered frompressurized packs. Drops can be delivered via a simple eyedropper-capped bottle, or via a plastic bottle adapted to deliver liquidcontents dropwise, via a specially shaped closure.

Further, in some embodiments, the extended release pharmaceuticalformulation comprising one or more compounds in the sialic acidbiosynthetic pathway or derivative thereof may also be used incombination with other therapeutic agents.

Methods of Making Extended Release Formulations

Methods of making extended release formulations detailed herein are alsoprovided. In one aspect, the formulation components (which mayoptionally be delumped and sieved to a desired range of particle size)are combined and mixed to provide a uniform formulation blend, which mayfurther be used to prepare particular dosage forms, such as tablets orcapsules, e.g., for oral administration. Particular dosage forms, onceprepared, may be further modified to provide the final drug product,such as, e.g., by administering a coating to a tablet formed from anextended release formulation blend. Preparation of the extended releaseformulations may be accomplished through known techniques, such asdirect compression, dry granulation and wet granulation.

Direct compression may be accomplished by delumping the formulationcomponents and sieving to a desired range of particle size, which may bethe same or different size for individual formulation components. Thecomponents are then blended, which may be accomplished by one or aseries of blending steps until all formulation components are blended.The blended formulation may, if desired, be direct compressed to providethe desired product, which may be in the form of a dosage suitable fororal administration, such as a tablet. The blended formulation may alsobe filled into capsules or other forms for solid-dosage administration,e.g., for oral administration.

Dry granulation may also be utilized to prepare the extended releaseformulations detailed herein, and may be used to improve the flow orother characteristic of a blend of formulation components to be formedinto a final drug product. One example of dry granulation includesdelumping and/or sieving the formulation components, blending theformulation components and feeding the blend through, e.g., a rollercompactor that produces a ribbon of compressed product, then milling theresulting ribbon. The milled product may then be compressed as detailedabove or further blended with additional formulation components andcompressed.

Wet granulation may also be utilized to prepare the extended releaseformulations. For example, the formulation components may be delumpedand sieved to the desired size, and blended. The resulting blend may beadded to an appropriate fluid bed processor equipped with a spray gunfor fluidizing the blended formulation components using standardpractices. The resulting granulation is dried (e.g., in the fluid bed)and milled to a desired range of particle sized and may be used forpreparation of a final formulation. Wet granulation may also utilizehigh shear wet granulation (blended components are mixed, and frequentlychopped while the solvent, typically water or other aqueous-basedsolvent, is sprayed over the mass during granulation).

Extended release formulations that are in tablet form preferably arecompressed to a sufficient hardness to prevent premature ingress of amedium (e.g., aqueous medium) and to prevent surface pitting andbreakage during coating, when applicable.

It is understood that extended release formulation blends are provided,such as a final formulation blend comprising a therapeutic agent and allformulation components in a final product (e.g., a blend comprising atherapeutic agent, a polymer, an excipient and a lubricant) as well asintermediate formulation blends that contain a portion of allformulation components in a final product (e.g., a blend comprising atherapeutic agent and a polymer but not an excipient or a lubricant,where the final product contains an excipient and a lubricant).

Methods of Treating and Preventing Sialic Acid Deficiencies

Provided herein are also methods of treating and/or preventing sialicacid deficiencies in an individual in need thereof by administering aneffective amount of one or more compounds in the sialic acid pathway orderivatives thereof in any extended release formulation describedherein. The methods may comprise administration of an effective amountof any of the formulations detailed herein, including any of theformulations under the heading “Extended Release Formulations,”including but not limited to any of the formulations of Tables A-E.Thus, although certain formulations are detailed below, it is understoodthat any extended release formulations described herein may be employedin any of the methods provided herein. In one varation, the extendedrelease formulation comprises sialic acid, or a pharmaceuticallyacceptable salt thereof, as the therapeutic agent. In another variation,the extended release formulation comprises MaNAc, or a pharmaceuticallyacceptable salt thereof, as the therapeutic agent. In another variation,the extended release formulation comprises sialic acid, or apharmaceutically acceptable salt thereof and MaNAc, or apharmaceutically acceptable salt thereof, as the therapeutic agents. Inanother variation, the extended release formulation comprises a prodrugof one or more compounds in the sialic acid biosynthetic pathway such asa prodrug of sialic acid, or a pharmaceutically acceptable salt thereof,as the therapeutic agent. In another variation, the extended releaseformulation comrpises sialic acid, or a pharmaceutically acceptable saltthereof, as the therapeutic agent and further comprises a hydocolloidpolymer, an anionic, pH-dependent gel forming co-polymer and a waterswellable, pH independent polymer and optionally further comprises alubricant and/or an excipient. In a particular variation, the extendedrelease formulation comprises sialic acid, or a pharmaceuticallyacceptable salt thereof, carrageenan, sodium alginate and eitherhypromellose or polyethylene oxide. In a further variation, the extendedrelease formulation comprises sialic acid, or a pharmaceuticallyacceptable salt thereof, carrageenan, sodium alginate, eitherhypromellose or polyethylene oxide, magnesium strearate andmicrocrystalline collulose and colloiddal silicon dioxide. In oneaspect, the extended release formulation is a formulation of Table E. Inyet another aspect, the extended release formulation is a formulation ofTable 8. In some embodiments, the one or more compounds in the sialicacid pathway or derivatives thereof include ManNAc or a derivativethereof and/or sialic acid or a derivative thereof. For example,provided herein are methods of treating and/or preventing sialic aciddeficiencies in an individual in need thereof by administering aneffective amount of ManNAc and sialic acid in any extended releaseformulation described herein. In some embodiments, the methods oftreating and/or preventing sialic acid deficiencies increase sialic acidproduction. In some embodiments, the methods of treating and/orpreventing sialic acid deficiencies increase sialylation of effectedtissue. In some embodiments, the method of treating and/or preventingsialic acid deficiencies comprises administering an extended releaseformulation comprising a drug load of about 30-60% (e.g., sialic acidand/or ManNAc), about 20-30% w/w hypromellose (e.g. hypromellose Type2208 or Methocel K100M), about 20-25% w/w sodium alginate (e.g.Protanal), about 1-5% w/w lamda carrageenan (e.g. Viscarin GP-209),about 1-10% w/w of microcrystalline cellulose and colloidal silicondioxide (e.g., Prosolv® SMCC HD90), about 0.1-1% w/w/magnesium stearate(e.g. HyQual®), and about 1-5% of an enteric coating (e.g. Opadry® IIWhite). In some embodiments, the method of treating or preventing sialicacid deficiencies comprises administering an extended releaseformulation comprising a drug load of about 30-60% (e.g., sialic acidand/or ManNAc), about 20-30% w/w polyethylene oxide (e.g. Polyox WSR),about 20-25% w/w sodium alginate (e.g. Protanal), about 1-5% w/w lamdacarrageenan (e.g. Viscarin GP-209), about 1-10% w/w of microcrystallinecellulose and colloidal silicon dioxide (e.g., Prosolv® SMCC HD90),about 0.1-1% w/w/magnesium stearate (e.g. HyQual®), and about 1-5% of anenteric coating (e.g. Opadry® II White).

Provided herein are also methods of increasing production of sialic acid(e.g., increasing production of sialic acid in muscle tissue) and theproximate substrate for glycosylation, CMP-sialic acid in an individualin need thereof by administering an effective amount of one or morecompounds in the sialic acid pathway or derivatives thereof in anyextended release formulation described herein. In some embodiments, theone or more compounds in the sialic acid pathway or derivatives thereofinclude ManNAc or a derivative thereof and/or sialic acid or aderivative thereof. For example, provided herein are methods ofincreasing production of sialic acid (e.g., increasing production ofsialic acid in muscle tissue) in an individual in need thereof byadministering an effective amount of ManNAc and sialic acid in anyextended release formulation described herein.

Provided herein are also methods of increasing sialylation of muscletissue in an individual in need thereof by administering an effectiveamount of one or more compounds in the sialic acid pathway orderivatives thereof in any extended release formulation describedherein. In some embodiments, the one or more compounds in the sialicacid pathway or derivatives thereof include ManNAc or a derivativethereof and/or sialic acid or a derivative thereof. For example,provided herein are methods of increasing sialylation of muscle tissuein an individual in need thereof by administering an effective amount ofManNAc and sialic acid in any extended release formulation describedherein.

Provided herein are also methods of improving muscle function in anindividual in need thereof by administering an effective amount of oneor more compounds in the sialic acid pathway or derivatives thereof inany extended release formulation described herein. In some embodiments,the one or more compounds in the sialic acid pathway or derivativesthereof include ManNAc or a derivative thereof and/or sialic acid or aderivative thereof. For example, provided herein are methods ofimproving muscle function in an individual in need thereof byadministering an effective amount of ManNAc and sialic acid in anyextended release formulation described herein.

Sialic acids are important for proper development and functioning ofmany organs and tissues, and a deficiency of sialic acid can give riseto many different types of diseases and conditions. Other types ofmuscle diseases have also shown that glycosylation is important formuscle function. Nishino and Ozawa, Curr. Opin. Neurol. 15:539-544(2002). In some embodiments, the sialic acid deficiency is a myopathy,muscular atrophy and/or muscular dystrophy. Myopathies that can betreated with the present compositions and methods also include distalmyopathy with rimmed vacuoles (Nonaka myopathy) and the musculardystrophy hereditary inclusion body myopathy (HIBM). In someembodiments, the methods of treating and/or preventing increasesialylation of muscle tissue. In some embodiments, the methods oftreating and/or preventing improve muscle function and reduce muscleinjury from physical activity, as measures by creatine kinase plasmalevels after exercise. In some embodiments, the methods of treating orpreventing muscle dysfunction will improve independent ambulation, stairclimbing, foot drop, getting up from a chair and walking, hand grip andmanipulation and pulmonary function. In some embodiments, the methodfurther comprises identifying an individual in need thereof bydetermining genotype or expression levels of the gene GNE.

In some embodiments, the sialic acid deficiency is a kidney conditionand diseases (e.g., those involving proteinuria and hematuria).Proteinuria involves leakage of protein from the blood into the urine.If the amount of protein in the urine is very high, this condition isoften called nephrotic syndrome. Several types of diseases exhibit thesymptoms of proteinuria, including high blood pressure, infections,reflux nephropathy, diabetes, and various types of glomerulonephritis,including minimal change nephrosis. Hematuria simply means blood in theurine (e.g., gross hematuria or microscopic hematuria). In someembodiments, the methods of treating and/or preventing increasesialylation of kidney tissue.

In embodiments of any of the methods, a therapeutically effective amountof one or more compounds in the sialic acid pathway or derivativesthereof is provided over a period of greater than about any of 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, or 24 hours. In some embodiments, a therapeutically effective amountof one or more compounds in the sialic acid pathway or derivativesthereof is provided over a period of greater than about 12 hours orgreater than about 24 hours. In embodiments of any of the methods, atherapeutically effective amount of one or more compounds in the sialicacid pathway or derivatives thereof is provided over a period of betweenabout any of 1-24 hours, 4-24 hours, 6-24 hours, 8-24 hours, or 12-24hours. In embodiments of any of the methods, a therapeutically effectiveamount of one or more compounds in the sialic acid pathway orderivatives thereof is provided over a period of about any of 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, or 24 hours. In some embodiments, a therapeutically effective amountof one or more compounds in the sialic acid pathway or derivativesthereof is provided over a period of about 12 hours or about 24 hours.In some embodiments, the therapeutically effective amount is provided tothe bloodstream of the individual. In some embodiments, thetherapeutically effective amount is provided to muscle tissue of theindividual. In some embodiments, the one or more compounds in the sialicacid pathway or derivatives thereof include ManNAc or a derivativethereof and/or sialic acid or a derivative thereof. For example, in someembodiments, a therapeutically effective amount of ManNAc and/or sialicacid is provided to muscle tissue of the individual over a period ofbetween about any of 1-24 hours, 4-24 hours, 6-24 hours, 8-24 hours, or12-24 hours.

In embodiments of any of the methods, the individual in need thereof isprovided a substantially constant (i.e., without large burst of drugavailability and deficiencies in drug availability to the blood and/ortissues of interest (e.g., muscle tissue)) therapeutically effectiveamount of one or more compounds in the sialic acid pathway orderivatives thereof over a period of greater than about any of 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, or 24 hours. In embodiments of any of the methods, the individual inneed thereof is provided a substantially constant therapeuticallyeffective amount of one or more compounds in the sialic acid pathway orderivatives thereof over a period of between about any of 1-24 hours,4-24 hours, 6-24 hours, 8-24 hours, or 12-24 hours. In embodiments ofany of the methods, the individual in need thereof is provided asubstantially constant therapeutically effective amount of one or morecompounds in the sialic acid pathway or derivatives thereof over aperiod of about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours. In some embodiments,the one or more compounds in the sialic acid pathway or derivativesthereof include ManNAc or a derivative thereof and/or sialic acid or aderivative thereof. For example, in some embodiments, the individual inneed thereof is provided a substantially constant therapeuticallyeffective amount of ManNAc and/or sialic acid to muscle tissue of theindividual over a period of between about any of 1-24 hours, 4-24 hours,6-24 hours, 8-24 hours, or 12-24 hours.

In embodiments of any of the methods, the individual in need thereof isprovided a therapeutically effective amount of one or more compounds inthe sialic acid pathway or derivatives thereof with a T_(max) of betweenabout any of 2-4 hours, 3-4 hours, 6-8 hours, 6-12 hours, 6-15 hours,12-18 hours, or 18-24 hours. In some embodiments, the one or morecompounds in the sialic acid pathway or derivatives thereof includeManNAc or a derivative thereof and/or sialic acid or a derivativethereof. In some embodiments, the one or more compounds in the sialicacid pathway or derivatives thereof include ManNAc and/or sialic acid.

In embodiments of any of the methods, less than about any of 10%, 20%,30%, 40%, 50%, 60%, or 70% of one or more compounds in the sialic acidpathway or derivatives thereof is excreted from the individual after onehour. In embodiments of any of the methods, less than about any of 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of one or more compounds inthe sialic acid pathway or derivatives thereof is excreted from theindividual after four hours. In some embodiments, the one or morecompounds in the sialic acid pathway or derivatives thereof includeManNAc or a derivative thereof and/or sialic acid or a derivativethereof. In some embodiments, the one or more compounds in the sialicacid pathway or derivatives thereof include ManNAc and/or sialic acid.

In embodiments of any of the methods, the one or more compounds in thesialic acid pathway or derivatives thereof are administered to anindividual in need thereof between about any of 0.1-50 g/day, 0.5-25g/day, 1-15 g/day, 1-10 g/day, 2-5 g/day, 0.2-25 g/day, 0.3-12 g/day,0.4-10 g/day, 0.5-8 g/day, and 0.7-6 g/day. In some embodiments, the oneor more compounds in the sialic acid pathway or derivatives thereof areadministered between about 2 g/day and 5 g/day. In embodiments of any ofthe methods, the one or more compounds in the sialic acid pathway orderivatives thereof are administered to an individual in need thereofbetween about any of 0.01-750 mg/kg, 0.5-500 mg/kg, 1-250 mg/kg, 2.5-100mg/kg, or 5-50 mg/kg. In some embodiments, the one or more compounds inthe sialic acid pathway or derivatives thereof are administered to anindividual in need thereof between about 5 mg/kg and 50 mg/kg. In someembodiments, the one or more compounds in the sialic acid pathway orderivatives thereof include ManNAc or a derivative thereof and/or sialicacid or a derivative thereof. For example, in some embodiments, ManNAcand/or sialic acid are administered to an individual in need thereofbetween about 5 mg/kg and 50 mg/kg.

In embodiments of any of the methods, the one or more compounds in thesialic acid pathway or derivatives thereof are administered to anindividual in need thereof between about any of 0.01-750 mg/kg/day,0.5-500 mg/kg/day, 1-250 mg/kg/day, 2.5-100 mg/kg/day, or 5-50mg/kg/day. In some embodiments, the one or more compounds in the sialicacid pathway or derivatives thereof are administered to an individual inneed thereof between about 5 mg/kg/day and 50 mg/kg/day. In someembodiments, the one or more compounds in the sialic acid pathway orderivatives thereof include ManNAc or a derivative thereof and/or sialicacid or a derivative thereof. For example, in some embodiments, ManNAcand/or sialic acid are administered to an individual in need thereofbetween about 5 mg/kg/day and 50 mg/kg/day.

In some embodiments, the effective amount of one or more compounds inthe sialic acid pathway or derivatives thereof in any extended releaseformulation is administered once a day, twice a day, three times a day,or four times a day.

The amount of the extended release formulation according to anembodiment of the invention to be administered to a human body may beappropriately selected in accordance with the absorption rate in thebody, rate of inactivation, rate of excretion, the age, gender andcondition of the patient, severity of the disease, or the like. Suchfactors can be readily determined by the clinician employing animalmodels or other test systems that are available in the art.

Administration of the therapeutic agents in accordance with the presentinvention may be in a single dose, in multiple doses, in a continuous orintermittent manner, depending, for example, upon the recipient'sphysiological condition, whether the purpose of the administration istherapeutic or prophylactic, and other factors known to skilledpractitioners. The administration of one or more compounds in the sialicacid pathway or derivatives thereof may be essentially continuous over apre-selected period of time or may be in a series of spaced doses. Bothlocal and systemic administration is contemplated.

Unit Dosages and Articles of Manufacture

Also provided herein are articles of manufacture and unit dosages whichinclude the extended release formulations comprising one or morecompounds in the sialic acid pathway or derivatives thereof describedherein.

Provided herein are articles of manufacture or kits comprising: (a) acontainer comprising the extended release pharmaceutical formulationcomprising one or more compounds in the sialic acid biosynthetic pathwayor derivative thereof described herein; and (b) a package insert withinstructions for treating and/or preventing a sialic acid deficiency ina patient. In some embodiments, the one or more compounds in the sialicacid pathway or derivatives thereof include ManNAc or a derivativethereof and/or sialic acid or a derivative thereof.

The article of manufacture comprises a container and a label or packageinsert on or associated with the container. Suitable containers include,for example, bottles, vials, syringes, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds or contains a formulation and may have a sterile access port (forexample the container may be an intravenous solution bag or a vialhaving a stopper pierceable by a hypodermic injection needle). At leastone active agent in the composition is the polypeptide. The label orpackage insert indicates that the composition's use in a subject withspecific guidance regarding dosing amounts and intervals of polypeptideand any other drug being provided. The article of manufacture mayfurther include other materials desirable from a commercial and userstandpoint, including other buffers, diluents, filters, needles, andsyringes. In some embodiments, the container is a syringe. In someembodiments, the syringe is further contained within an injectiondevice. In some embodiments, the injection device is an autoinjector.

A “package insert” is used to refer to instructions customarily includedin commercial packages of therapeutic products, that contain informationabout the indications, usage, dosage, administration, contraindications,other therapeutic products to be combined with the packaged product,and/or warnings concerning the use of such therapeutic products.

Provided herein are also unit dosages which include the extended releaseformulations comprising one or more compounds in the sialic acid pathwayor derivatives thereof. In some embodiments, the one or more compoundsin the sialic acid pathway or derivatives thereof include ManNAc or aderivative thereof and/or sialic acid or a derivative thereof

Unit dosage forms comprising any of the extended release formulationsdescribed herein, including but not limited to those formulationsdetailed under the heading “Extended Release Formulations,” such as anyof the formulations of Tables A-E, are described. These unit dosageforms can be stored in a suitable packaging in single or multiple unitdosages and may also be further sterilized and sealed. For convenienceand ease of patient compliance, the extended release formulations may bedelivered in the form of unit dosage forms, which may be administered toan individual. In one variation, the extended release formulation is asolid substance and unit dosage forms thereof may be prepared in theform of tablets, capsules, sachets and chewable tablets. In one aspect,the dosage form is in the form of a capsule or tablet, preferably in theform of a tablet.

The preparation of the unit forms generally involves a step of preparinga blend filling, either by volume or weight. For example, in productionof tablets and capsules, the extended release formulation blend isvolume filled into a die or capsule, respectively. In one aspect, abatch of unit dosage forms has the same potency (amount of drug per unitdosage form) within an allowable margin, which in one variation is arelative standard deviation (RSD) of less than 6% and in anothervariation is less than 8.0 or 7.8%.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed.

Example 1

Human myoblasts are obtained from an HIBM patient and grown anddifferentiated into myotubes. After an appropriate washout, the cellsare placed in protein free or sialic acid free medium and then treatedwith differing concentrations of sialic acid, ManNAc, or both fordifferent periods of time. At time points 0 (before supplementation), 2hours, 8 hours, 16 hours, and 24 hours after the start (and differenttimes after removal of replacement substrates from each culture),internal sialic acid, CMP-sialic, and glycosylation of newly synthesizedproteins are measured. Glycosylation replacement after 24 hours isevaluated and a time course is also determined for glycosylation withreplacement, runs out of substrate, and stops glycosylating.

Example 2

Human HIBM myoblasts are obtained and differentiated into myotubes asdescribed in Example 1 and this time, a matrix of sialic acid andN-acetylmannosamine is given for either short bursts of 1-2 hours orlonger periods of 4 and 8 hours. The onset of normalization ofsialylation and the peak efficiency is evaluated, as well as the time todecline of normal sialylation. Efficacy of single and combinationformulations in replacement treatment and effective time are determined.

Example 3

A large variety of human fibroblasts from HIBM patients are obtainedwith different mutations and clinical phenotypes. Each line is titratedto its 50% maximal correction with sialic acid and ManNAc independently.Replacement efficacy is evaluated for different lines from differentpatients.

Example 4

A mouse model of HIBM is treated with either standard sialic acid orManNAc or given both compounds together. An additional group is giventhese items in an extended release formulation. The mice are evaluatedusing the procedure described in Malicdan et al., Nat. Medicine15(6):690-695 (2009) for muscle strength and clinical outcome. Inaddition, mice are analyzed at different time points after a dose andduring treatment to assess glycosylation and intermediates in the muscletissues. Clinical effect and the best steady state restoration ofintermediates are evaluated.

Example 5

To assess the minimal concentration of sialic acid metabolite requiredto maintain sialylation optimally, myoblasts, myotubes or humanfibroblasts are cultured in sialic acid free medium until they reach anabnormal level of sialylation at steady state. A series ofconcentrations to these cultures are added and evaluated for therestoration of glycosylation. Concentration in the medium required atsteady state to replace the missing sialylation is determined. Thisconcentration provides a target for a minimum plasma concentrationwithin patients treated with an extended release formulation.

Example 6

Preparation of Sialic Acid 250 mg Strength Tablets Using Dry BlendMethod of Manufacture Experimental/Materials

Sialic Acid (Food & Bio Research center, Inc. Kyoto Japan) was stored inaluminum foil bags at −20 C. However, handling and processing ofprototypes were all under ambient room temperature. In-process materialsand bulk tablets were stored in double polyethylene bags with desiccant.The sialic acid was evaluated for physical properties consisting ofmorphology, particle size by sieve analysis, bulk and tap density.

50 gram lab-scale batches were prepared using bag-blending, manualfilling and hand turning of the tablet press to compress tablets toevaluate dissolution as the first level of screening. Tablets weremanufactured using the ProCR platform. Their formulas are listed belowin Table 1 and 2.

TABLE 1 Quantitative Formula for Sialic Acid, ProCR Hypromellose 250 mgTablets: Ingredient Vendor mg/Tablet % w/w g/batch Sialic Acid (N- Foodand 249.75 33.3 16.65 Acetylneuraminic acid) BioResearch Center, IncHypromellose, Type 2208 Colorcon 225.0 30.0 15.0 (Methocel ® K100 MPremium CR) Sodium Alginate FMC 187.5 25.0 12.5 (Protanal ® LF 120M)Biopolymer Carrageenan (Viscarin GP- FMC 37.5 5.0 2.5 209) BiopolymerMicrocrystallline Cellulose JRS Pharma 46.5 6.2 3.1 and ColloidalSillicon Dioxide (ProSolv ® SMCC HD 90) Magnesium Stearate Mallinckrodt3.75 0.5 0.25 (HyQual ®), Vegatable Source Product Code 2257 Total 750100% 50

TABLE 2 Quantitative Formula for Sialic Acid ProCR Polyox, 250 mgTablets: Ingredient Vendor mg/Tablet % w/w g/batch Sialic Acid (N- Foodand 249.75 33.3 16.65 Acetylneuraminic acid) BioResearch Center, IncPolyethylene Oxide WSR Dow 225.0 30.0 15.0 (Polyox) Chemical CompanySodium Alginate FMC 187.5 25.0 12.5 (Protanal ® LF 120M) BiopolymerCarrageenan (Viscarin GP- FMC 37.5 5.0 2.5 209) BiopolymerMicrocrystallline Cellulose JRS Pharma 46.5 6.2 3.1 and ColloidalSillicon Dioxide (ProSolv ® SMCC HD 90) Magnesium Stearate Mallinckrodt3.75 0.5 0.25 (HyQual ®), Vegatable Source Product Code 2257 Total 750100 50

Sialic Acid, hypromellose Type 2208, sodium alginate, carrageenan andmicrocrystalline cellulose with colloidal silicon dioxide were delumpedusing a #20 USA standard sieve and weighed per the quantitative formula.The ingredients were combined in a small ziplock bag and blended for 15minutes. Magnesium stearate was delumped using a #40 USA standardscreen, weighed per quantitative formula, and added to the blendedingredients in the bag. The ingredients were blended for an additionalthree minutes. The final blends, as well as the un-sieved sialic acidwere characterized using bulk density, tap density, particle size sieveanalysis, Carr's Compressibility Index, and minimum critical orifice.The final blend of each prototype was compressed on the Korsch PH100tablet press. The resulting tablets were submitted to the analytical labfor dissolution testing.

Sialic Acid Characterization

Sialic Acid was visually characterized as a white fluffy powderysubstance. Its bulk density was 0.293 g/mL, and its tap density was0.419 g/ml. The Carr's Compressibility Index was 30%, and the minimumcritical orifice diameter was 18 mm. The particle size sieve analysis ofSialic Acid (Table 3) revealed a distribution of coarse and midsizeparticles as shown in FIG. 2. The sialic aicd was sized prior toblending to facilitate blend homogeneity.

TABLE 3 Particle Size Distribution for Sialic Acid Unsieved Sialic Acid(N- Sieve # (Mesh size (um)) Acetylneuraminic acid) 20 (850) 34.16 40(425) 26.87 60 (250) 15.57 100 (150) 10.75 200 (75) 9.3 325 (45) 1.77Pan (<45) 1.45

ProCR Sialic Acid, 250 mg CR Tablets

Both prototype blends, ProCR hypromellose and ProCR Polyox, werecompressed into tablets using 0.3300×0.7100 inch modified oval toolingtargeting a tablet weight of 750 mg and a hardness range of 17 to 20 Kp.During tableting, powder bridging in the die cavity was observed forProCR hypromellose. This was an indication that the blend needed to bedensified to improve flowability on the tablet press. ProCR Polyoxappeared denser and seemed to flow better on the tablet press. However,its Carr's Compressibility Index and minimum critical orifice diameterresults, as shown in Table 4, indicated that it also needed furtherprocessing such as, granulation. The particle size distribution of thePolyox prototype seemed to be more dispersed over various screen sizesthan the hypromellose prototype shown in Table 5 and Figure

TABLE 4 Physical Characterization Results of Sialic Acid 250 mg Bulk TapCarr's Flodex Density Density Compressibility Critical Powder (g/mL)(g/mL) Index (%) Orifice (mm) Sialic Acid (N- 0.293 0.419 30   18Acetylneuraminic (poor flow) acid) Prototype 1 0.359 0.543 33.8 20(Hypromellose) (very poor flow) Prototype 2 0.439 0.716 38.7 18 (Polyox)(very very poor flow)

TABLE 5 Particle Size Distribution for ProCR Sialic Acid, 250 mgTablets. Prototype 1 with Prototype 2 with Sieve # HypromellosePolyethylene oxide (Mesh size (um)) % Retain % Retain 20 (850) 0.34 0.8440 (425) 16.02 15.20 60 (250) 9.2 12.88 100 (150) 10.3 15.49 200 (75)10.3 18.51 325 (45) 33.91 24.96 Pan (<45) 19.89 12.12

The compression of the tablets resulted in a weight range of 3-5% of thetarget of 750 mg. The variability was primarily due to the manualfilling and poor flow. Regardless of the weight variability, the tabletappearance and hardness was good, ranging from 13 to 18 Kp, as listed inTable 6. The dissolution results showed a first order sustained releaseprofile over a 12 hour period, as shown in Table 7 and FIG. 4.

TABLE 6 Physical Data of Sialic Acid 250 and 325 mg Tablets HypromellosePolyox Hypromellose Polyox 250 mg 250 mg 325 mg 325 mg Test TabletsTablets Tablets Tablets Tablet Weight (mg) 744-787 746-751 747-766745-771 Tablet Thickness (in) 0.268-0.271 0.261-0.263 0.291-0.2950.283-0.286 Tablet Hardness (kp 17.5 18.3 13.2 13.0 Tablet Friability(%) ND ND ND ND ND: Not determined

TABLE 7 Dissolution Results of Direct Compression PrototypesHypromellose Polyox Hypromellose Polyox 250 mg 250 mg 325 mg 325 mg TestTablets Tablets Tablets Tablets Dissolution (Average n = 3) % Release 2hr 26 30 33 35 4 hr 41 50 50 55 6 hr 53 66 67 71 8 hr 65 77 78 82 12 hr82 97 92 95 16 hr * — — 99 103 24 hr 100  112  — — * Represented as lasttime point in graph

Example 7 Preparation of Sialic Acid 325 and 500 mg DevelopmentPrototypes

Initially, two small 50 gram dry blend batches were manufactured with anincreased drug load from 33% w/w to 43% w/w to verify that the drugrelease profile was acceptable. The two compositions are listed in Table8 as hypromellose and Polyox. The tabletting was done as describedbefore using a manual fill into the die cavity.

TABLE 8 Quantitative Formula for Sialic Acid 325 mg and 500 mg sustainedrelease Tablets Prototypes: mg/Tablet mg/Tablet g/batch g/batch ProCRProCR 50 g 1800 g Ingredient Vendor Hypromellose Polyox % w/w size sizeSialic Acid (N- Food and 325.0 325.0 43.3 21.65 779.4 AcetylneuraminicBioResearch acid) Center, Inc Hypromellose, Colorcon 191.3 — 25.5 12.75459 Type 2208 (Methocel ® K100 M Premium CR) Polyethylene — 191.3 25.512.75 459 Oxide WSR (Polyox) Sodium Alginate FMC 159.0 159.0 21.2 10.60381.6 (Protanal ® LF Biopolymer 120M) Carrageenan FMC 31.5 31.5 4.2 2.1075.6 (Viscarin Biopolymer GP-209) Microcrystallline JRS Pharma 39.8 39.85.3 2.65 95.4 Cellulose and Colloidal Sillicon Dioxide (ProSolv ® SMCCHD 90) Magnesium Mallinckrodt 3.8 3.8 0.5 0.25 9.0 Stearate (HyQual ®),Vegatable Source Product Code 2257 Total for 325 mg Strength 750.4 750.4100 50 1800 Total for 500 mg Strength 1154.5 1154.8 100 — —

Wet Granulation Method of Manufacture

In order to avoid bridging and poor flow during compression batch sizeswere scaled up from 50 grams to 1800 grams, and a high shear granulationmethod of manufacture was used to produce 325 and 500 mg dose strengthswhile maintaining good tablet compression properties. The 325 and 500 mgdose strengths shared a common blend that was divided prior tocompression. Two tablet sizes were produced: A 325 mg dose strengthtablet with a length of 17.7 mm, a width of 9.1 mm and a thickness of6.7 mm; and a 500 mg dose strength tablet with a length of 19.3 mm, awidth of 9.7 mm and a thickness of 8.0 mm). The following equipment andprocess were used to make these tables.

Experimental/Materials

All raw materials were used as received from vendors as listed in Table8. The batch size was 1800 grams. The following equipment was used:

-   -   Fielder PP1 High Shear Granulator    -   Niro-Aeromatic MP-1 Multi-processor    -   FitzMill JT Homoloid equipped with knives forward, 0.079″ round        hole screen 4 Qt PK Blender    -   Korsch PH100 tablet Press equipped with 0.350″×0.6875″ modified        oval tooling for the 750 mg tablet and 0.374″×0.7480″ modified        oval tooling for the 1154 mg tablet Accela-cota model 24MK III        (24″ coating pan)

All the raw materials with exception of magnesium stearate were chargedto the PP-1 granulator and premixed for 3 minutes at 300 rpm impellerspeed, no chopper. A baseline loss on drying determination was performedand the ungranulated hypromellose formula was determined to be 3.4%water while the Polyox formulation was 2.9%. Water was sprayed atapproximately 200 grams/minute while mixing at 300 rpm with a slowchopper speed. The hypromellose formulation used 43% water (778 g watersprayed) of the 1.8 kg batch size while the Polyox formulation sprayed52% water (905 g water sprayed) with a 2 minute post spray mix. Thegranulation was transferred into the MP-1 fluid bed and dried with aninlet temperature of 75° C. until the loss on drying (LOD) was ≦3%;equal to or slightly lower than the baseline moisture of theun-granulated formulations. The dried granulation was passed through a#4 mesh hand screen. The large granules retained on the #4 mesh weresegregated and discarded. The remaining granules were sized through theFitzMill at low speed, knives forward. The blend was then lubricatedwith the magnesium stearate for 3 minutes. The final blend wascompressed into tablets using a Korsch rotary press. After dissolutionresults were obtained, the core tablets were coated with anon-functional, Opadry II, white to a weight gain of approximately 4.5%w/w.

Outline of Dissolution Conditions were as follows:

-   -   900 mL dissolution medium: 50 mM Phosphate, pH 6.8

100 RPM Baskets

37° C.

Time points: 2, 4, 6, 8, 12, 16 or 24 hours

The blending and granulation of the hypromellose based formulationproceeded smoothly. The hypromellose formulation processed well,producing a final blend with excellent flow that compressed well on thetablet press. The yield was excellent (96%) for a small scale batchsize.

The Poly Ethylene Oxide (Polyox) based formulation did not granulate aseasily. The Polyox formulation was over-granulated. The over-granulationcan be alleviated in the future by spraying less granulation water at aslower rate. An appreciable amount of the granulation was lost when thepartially dried granulation was screened through a 4 mesh sieved toremove large over-granulated agglomerates that resisted drying in thefluid bed. As a result, the batch yield was poor at 83%. The portion ofthe batch that was retained produced an excellent final blend, however.It flowed and compressed well on the tablet process and produced goodquality tablets. Polyox is known for being difficult to granulate sothis is not entirely unexpected. However, with the proper granulationparameters an excellent granulation can be attained.

Physical data for sialic acid 325 mg final blends, sialic acid 325 mgtablets, and sialic acid 500 mg tablets are shown in Tables 9, 10 and11, respectively. Analytical results for sialic acid 325 and 500 mgtablets (uncoated) are shown in Table 12.

TABLE 9 Physical data for Sialic Acid, 325 mg Final Blends: (ProCRHypomellose) (ProCR Polyox) Sieve # (% Retain) Mesh size (um) 14 (1400)0.10 1.32 30 (600) 42.89 45.4 40 (425) 12.28 14.39 140 (106) 33.98 29.89200 (75) 5.42 3.24 325 (45) 4.61 4.86 Pan (<45) 0.72 0.91 Blend BulkDensity (g/mL) 0.54.9 0.54.5 Tap Density (g/mL) 0.646 0.619 %Compressibility 15 12 Flowdex 10 6

TABLE 10 Physical Data of Sialic Acid 325 mg Tablets at VariousHardnesses Formulation A Formulation B Formulation C Formulation DFormulation E Formulation F Test Hypromellose Polyox Tablet HardnessLevel Low Medium High/max Low Medium High/max Ave. Weight (mg) 759.5 754754 739 745 750 Ave. Thickness (in) 0.279 0.270 0.259 0.260 0.247 0.253Ave. Hardness (kp) 6.5 10.0 14.4 9.5 17.9 15.7 Ave. Friability (%)Failed 0.2 0.1 0.1 0.0 0.2 Note: Average of 10 tablets

TABLE 11 Physical Data of Sialic Acid 500 mg Final Blends and TabletsFormulation G Formulation H Formulation I Formulation J Formulation KFormulation L Test Hypromellose Polyox Bulk Density (g/mL) 0.55 0.54Tablet Hardness Level Low Medium * High/max Low Medium High/max Ave.Weight (mg) 1170 ND 1152 1158 1154 1160 Ave. Thickness (in) 0.324 ND0.315 0.310 0.307 0.297 Ave. Hardness (kp) 11.3 ND 13.2 12.9 14.0 20.2Ave. Friability (%) 0.2 ND 0.0 0.1 0.0 0.0 Note: Average of 10 tablets *ND: not determined

TABLE 12 Analytical Results or Sialic Acid 325 mg and 500 mg SR Tablets(Uncoated) Formulation Formulation Formulation Formulation C F I K TestHypromellose Polyox Hypromellose Polyox Tablet Strength 325 325 500 500(mg) Assay (% LC) 96.3 97.8 Impurities (%) Total: 0.2 Total: 0.2Dissolution (Average n = 3) % Release 2 hr 34 34 29 29 4 hr 53 55 46 476 hr 67 72 59 62 8 hr 78 85 69 74 12 hr 91 96 84 89 24 hr 100 99 101 100

The dissolution results (FIG. 5) showed a first order sustained releaseprofile over a 12 hour period for both dose strengths and for both ProCRhypromellose and ProCR Polyox. Additionally, these results indicate thatthe dose proportional approach was successful in providing doseflexibility using a common blend at 750 and 1154 tablet final weights.

Example 8

Coating for Sialic Acid 325 and 500 mg SR Tablets ProCR Hypromellose andProCR Polyox Method of Manufacture

Eight kilograms of core tablets (approximately 1.5 kg of active tabletscombined with 6.5 kg of “sham” placebos to provided volume) were chargedinto an Accela-Cota coating equipment equipped with a 24″ coating panand two spray guns. The non-functional film coat was Opadry-II White(Colorcon Corporation formula Y-22-7719) at a 20% solids concentration.The purpose of the film coat was to improve aesthetics and in the futurefacilitate patient compliance for swallowing of the tablet. The targetend-point was 3-5% weight gain.

The coating process parameters were as follows:

Pan speed: Target 12-16 rpm

Inlet temperature: 70-85° C.

Outlet temperature: 39-42° C.

Bed temperature: 33-45° C.

Atomization pressure: 40 psi

Spray Rate: 50-60 g/min

Airflow: approximately 200 cfm

Gun to bed distance: 5″

The tablets coated well with no difficulties. Approximately 4% weightgain of coating was sufficient to provide good coverage of the tabletcores.

Prototype Stability

The white film coated tablets of Sialic Acid prepared using ProCRhypromellose and ProCR Polyox at 325 mg and 500 mg dose strengths werepackaged in thirty (30) units per bottle, one MiniPax desiccant, no coiland induction sealed using a Lepak Jr™ induction cap sealing system.Table 13 lists the packaging components used. All the acceptable tabletswere packaged and placed on a 12 month prototype stability program underICH conditions testing the stability at both 25° C. and 60% relativehumidity (RH) and 40° C. and 75% RH at 0, 1, 3, 6, and 12 months. Thetablets have been tested and monitored with respect to appearance,dissolution, moisture, assay and related substances, and initialstability results are shown in Table 14. The dissolution profile for thecoated 325 mg and 500 mg tablets is shown in FIG. 6.

TABLE 13 List of Packaging Components Component Material Description AAIRM # Bottle 100 cc Round White HDPE (38/400) PC-3714 Closure 38 mm CRC wFoil Seal MI Liner PC-3982 Desiccant MiniPax w 1.00 g Silica Gel-PacketPC-2637

TABLE 14 Analytical Results of Sialic Acid 325 mg and 500 mg SR Tablets(Coated), Initial Stability Formulation Formulation FormulationFormulation C F I K Test Hypromellose Polyox Hypromellose Polyox TabletStrength 325 325 500 500 (mg) % Moisture by 1.0 3.3 2.0 3.7 Karl FischerContent Uniformity 100.0 95.6 99.8 98.6 (Ave, n = 10) % RSD 1.5 2.5 1.52.6 AV 3.5 9.2 3.6 6.3 Assay (% LC) 100.6 97.8 98.8 96.9 Impurities (%)Total: <0.10 Total: <0.10 Total: <0.10 Total: <0.10 Dissolution (Ave. %Release, n = 6)) 2 hr 31 34 27 27 4 hr 50 54 42 43 6 hr 65 70 55 56 8 hr75 83 65 68 12 hr 90 96 79 83 24 hr 102 100 98 96

The formulation development activities successfully identified twodistinct sustained release prototypes for Sialic Acid in 325 and 500 mgdose strengths. The in-vitro dissolution release profile exhibited afirst order release over 12 hours in aqueous medium and pH of 6.8. Thesustained release ProCR platform was employed. This unique combinationof inert polymers provides a robust formulation that is pH independentand lends itself to granulation processes without affecting thedissolution release profile. This was the case for Sialic Acid 325 and500 mg dose strength SR tablets where a wet granulation process wasfound necessary to achieve densification and good tabletcompressibility.

With regard to chemical stability Sialic Acid 325 and 500 mg ProCRhypromellose and ProCR Polyox SR tablets showed acceptable assay,dissolution and content uniformity and easily passed USP testingcriteria. These prototypes are monitored through a 12 month ICHstability study.

As shown in FIGS. 5 and 6, the dissolution profiles of Sialic Acid ProCRhypromellose and ProCR Polyox uncoated and coated tablets areconsistent. There is no significant change in the sustained releaseprofile over the 12 hour release with the application of Opdary® IIWhite film coat. The analytical results for assay and related substancesare acceptable which indicates that the wet granulation, drying andcoating processes have no impact on the chemical integrity of the drug.

Example 9 Preparation of ManNAc 325 mg Development Prototypes

The ManNAc title formulation was prepared according to the methoddetailed above for Sialic Acid. The dissolution profile of ManNAc 325 mgTablets is shown in FIG. 7.

Core Tablet Results

Assay % LC = 93.5% Impurity % RS Sialic Acid <0.10% Sodium Pyruvate<0.10% N-Acetyl-D-Glucosamine 0.4% Acetic Acid <0.10% Total 0.4% KF Prep% water 1 3.5 2 3.3 Mean(2) 3.4 Content Uniformity Unit % LC 1 93.7 294.6 3 92.8 4 92.8 5 94.6 6 92.9 7 96.0 8 95.4 9 92.5 10 91.5 Mean (10)93.7 % RSD 1.5 SD 1.42735186 AV 8.2

Example 10 Pharmacokinetics of Sialic Acid Formulations Following aSingle Oral or Intravenous Dose in Male Dogs

The objective of this study was to evaluate the pharmacokinetics ofsialic acid following single oral or intravenous dose in male dogs.

A total of six male beagle dogs (Canis familiaris), originally fromBeijing Marshall Biotechnology Co., Ltd., were obtained from the PCS-SHGcolony and subjected to a general physical examination to ensure normalhealth status before study initiation. All animals were consideredsuitable for use and each animal was uniquely identified by a permanentskin tattoo number and/or letter on the ventral aspect of one pinna. Anacclimation period of five days was allowed between animal transfer andthe start of treatment in order to accustom the animals to thelaboratory environment.

Before dosing initiation, all animals were weighed and assigned totreatment groups. At the start of treatment, animals were 7-16 months ofage and ranged in weight as 6.4 to 9.4 kg. Animals were housedindividually in stainless steel cages equipped with a mesh-type floorand an automatic watering valve. A standard certified pelletedcommercial dog food (approximately 400 g of Certified Canine Diet 5C07,PMI Nutrition International, Inc.) was provided to each animal oncedaily, except during designated procedures. Maximum allowableconcentrations of contaminants in the diet (e.g., heavy metals,aflatoxin, organophosphates, chlorinated hydrocarbons, PCBs) werecontrolled and routinely analyzed by the manufacturers. It wasconsidered that there were no known contaminants in the food that couldhave interfered with the objectives of the study. Municipal tap water,which was softened, purified by reverse osmosis and exposed toultraviolet light, was freely available except during designatedprocedures. It was considered that there were no known contaminants inthe water that could have interfered with the objectives of the study.Each Animal was provided with a floor toy, except during designatedactivities.

The study design was as shown in Table 15:

TABLE 15 Experimental Design Dose Number Dose Dose Group Study TestLevel^(#) of Tablets Volume Concentration Animal No. Day ArticleTreatment^(a) (mg/kg) per animal (mL/kg) (mg/mL) Number 1 1 TA-1 PO — 10— — 3 (capsule) 6 TA-6 IV 25 — 0.5 50 9 TA-2 PO — 10 — — (tablet) 13TA-3 PO — 10 — — (tablet) 2 1 TA-1 PO — 10 — — 3 (capsule) 6 TA-6 IV 25— 0.5 50 9 TA-4 PO — 10 — — (tablet) 13 TA-5 PO — 10 — — (tablet) — =not applicable. ^(#)Dose level was expressed as free form. ^(a)Animalswere fasted overnight for approximately 16 hours prior to each dose andwere fed immediately after the 6 hr timepoint.

The first day of dosing was designated as Day 1. The subsequent dosingdays were Days 6, 9 and 13. On Days 1, 9 and 13, all animals were orallyadministered prepared capsules or tablets. On Day 6, all animalsreceived a single intravenous dose of TA-6 at 0.5 mL/kg. Each actualvolume of TA-6 administered was based on the most recent practical bodyweight of each animal. The test articles 1 through 6 are specified inTable 16.

TABLE 16 Specification of Test Articles Test Compositon/ Animal ArticleIdentification Concentration Dose Level Number TA-1 API in Capsule 325mg SA per 3250 mg SA per 101-103, Form capsule animal 201-203 TA-2Sialic Acid 325 mg SA/ 3250 mg SA per 101-103 Delayed 425 mg animalRelease Tablet excipient Formulation I (hypromellose) per tablet TA-3Sialic Acid 325 mg SA/ 3250 mg SA per 101-103 Delayed 425 mg animalRelease Tablet excipient Formulation II (polyethylene oxide) per tabletTA-4 Sialic Acid 500 mg SA/ 5000 mg SA per 201-203 Delayed 650 mg animalRelease Tablet excipient Formulation III (hypromellose) per tablet TA-5Sialic Acid 500 mg SA/ 5000 mg SA per 201-203 Delayed 650 mg animalRelease Tablet excipient Formulation IV (polyethylene oxide) per tabletTA-6 Sialic Acid IV 50 mg/mL 25 mg/kg SA 101-103, Formulation 201-203

Individual body weights were measured once during the predose period andprior to each dose on dosing days. There were no treatment-relatedclinical signs observed during the study period and no treatment relatedchanges in body weight or body weight gains noted for any animal duringthe study period.

Blood samples were collected into serum separate tubes from all animalson Days 1, 6, 9, 13 for processing to serum at the following timepoints: predose, 2 minutes (i.v. only), 5 minutes (i.v. only), 10minutes (i.v. only), 15 minutes, 30 minutes, 1, 2, 4, 6, 8 and 24 hourspostdose. Urine samples were collected into jars on wet ice or ice packsfrom all animals on Days 1, 6, 9, 13 at the following time intervals:predose (overnight for approximate 15 hours), 0 to 4, 4 to 8, 8 to 12hours postdose. Samples were collected according to Table 17 and Table18:

TABLE 17 PK Sample Collection Schedule Sample Collection Time PointsGroup (Time Post Dose) on Days 1, 6, 9 and 13 No. 0 min^(a) 2 min 5 min10 min 15 min 30 min 1 hr 2 hr 4 hr 6 hr 8 hr 24 hr 1 X X (IV only) X(IV only) X (IV only) X X X X X X X X 2 X X (IV only) X (IV only) X (IVonly) X X X X X X X X X = sample collected ^(a)Samples were collectedbefore dosing.

TABLE 18 Urine Sample Collection Schedule Sample Collection Time PointsGroup (Time Post Dose) on Days 1, 6, 9 and 13 No. Overnight^(a) 0-4 hr4-8 hr 8-12 hr 1 X X X X 2 X X X X X = sample collected ^(a)Blank urinewere collected overnight (approximate 15 hours) before each dose.

Blood samples were placed at room temperature for at least 30 minutesbut no more than 1 hour to clot prior to refrigerated centrifugation(approximately 4° C.) at approximately 2700 rpm for 10 minutes. Theserum separated from each sample was transferred into polypropylenetubes and placed on dry ice until transferred to a freezer (set tomaintain −80° C.). Urine samples were stored in a freezer (set tomaintain −80° C.) until analyzed.

Drug concentrations in serum and urine were determined by LC MS/MS usinga validated analytical procedure (Validation of a Method for theDetermination of Free Soluble Sialic Acid in Dog Serum and Urine byLiquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)-PN 102653;Long-term Matrix Stability Assessment of Free Soluble Sialic Acid in DogSerum and Urine by Liquid Chromatography-Tandem Mass Spectrometry(LC-MS/MS)-PN 102654. The method had a linear range from 10˜1000 μg/mLand the lower limit of quantitation was 10 μg/mL.

Data collection was performed using Analyst® from AB Sciex. Statisticalanalyses including regression analysis and descriptive statisticsincluding arithmetic means and standard deviations, accuracy andprecision were performed using Watson™ Laboratory Information ManagementSystem (LIMS) and Microsoft Excel.

Pharmacokinetic parameters were estimated using WinNonlin®pharmacokinetic software (Version 5.2.1, Pharsight Corp., Mountain View,Calif., USA). A non-compartmental approach consistent with theintravenous or oral route of administration was used for parameterestimation. All parameters were generated from individual sialic acidconcentrations in serum. Parameters were estimated using nominalsampling times relative to the start of each dose administration. Meanconcentrations were derived from 3 animals/group/time point forintravenous dosing occasion only. The actual timepoints were within therange of protocol specified. Serum concentration values obtained at thepredose time point were used as the concentration at time zero for oraldoses. The actual dose levels of test articles 1 through 5 werecalculated using the total amount of SA given to each animal based ontheir most practical body weight.

The area under the sialic acid individual serum concentration versustime curve (AUC) was calculated using the linear trapezoidal method withlinear interpolation. The terminal elimination phase of each individualconcentration versus time curve was identified using at least the finalthree observed concentration values. The slope of the terminalelimination phase was determined using log linear regression on theunweighted concentration data. The terminal elimination phase relatedparameters were not reported if the coefficient of determination wasless than 0.800, or the extrapolation of the AUC to infinity representedmore than 20% of the total area, or the terminal elimination phase couldnot be identified. The parameters described in Table 19 were observed orcalculated.

All data from serum including concentrations below LLOQ (except forthose below zero) were applied to pharmacokinetic analysis.

TABLE 19 Estimated Parameters from Serum Concentrations of Sialic AcidParameters Description of parameter Cmax The maximum observed arithmeticindividual concentration of sialic acid after dosing. Tmax The timeafter dosing when the maximum observed arithmetic individualconcentration of sialic acid was observed. AUC(0-t) The area under thesialic acid arithmetic individual concentration versus time curve fromtime zero to the time after dosing when the last quantifiableconcentration of the drug was observed. MRT(0-t) The mean residence timeof sialic acid estimated from time zero to the time after dosing atwhich the last quantifiable concentration of the drug was observedestimated or imputed by the linear or linear/log trapezoidal method. T½The apparent terminal elimination half life. AUC(0-inf) The area underthe arithmetic individual concentration versus time curve from time zeroto infinity. MRT(0-inf) The mean residence time estimated from time zeroto infinity. CL Clearance: the apparent volume of serum cleared of (IVonly) sialic acid per unit time following intravenous dosing. Clearancewas calculated for intravenous dose only. Vd The apparent volume ofdistribution of sialic acid, (IV only) determined from the terminalelimination phase following intravenous dosing. Volume of distributionwas calculated for intravenous dose only. Fs Absolute bioavailabilitybased on sialic acid levels in the serum following IV and oraladministration.

Urinary concentrations of sialic acid were subjected to calculationusing Microsoft® Excel, 2007. All data from urine includingconcentrations below LLOQ (except for those below zero) were applied.

The data of predose urine samples were applied to calculate the totalincrease in urinary excretion of sialic acid at 12 hours postdose. Theurinary excretion of sialic acid, as a percent of dose administered wasestimated for each dosing occasion. Based on the assumption that theamount of drug excreted in urine after oral administration was areflection of the dose absorbed, the bioavailability of sialic acid wasdetermined based on the percent excretion value following IV and oraladministration.

The parameters described in Table 20 were observed or calculated.

TABLE 20 Estimated Parameters from Urine Concentrations of Sialic AcidParameters Description of parameter Dose The amount of SA dosed peranimal contained in each test article. Mass The total mass of urinaryexcretion of sialic acid at Excreted 12 hours postdose. (0-12 hr) MassThe corrected value calculated by subtracting out Excreted thebackground masses based on predose data, (0-12 hr)- representing theincrease in urinary excretion of Corrected sialic acid at 12 hourspostdose. If corrected value was less than zero, the value was set tozero. Percent The corrected mass excreted of sialic acid as a Excretionpercent of dose administered. (0-12 hr) Fu Bioavailability based on thesialic acid levels in the urine following IV and oral administration.

There were no treatment-related clinical signs noted following eitheroral or intravenous administration of sialic acid over the study period.Skin red was noted for Animal Nos. 201 and 203 during the study, whichwas considered as incidental.

There were no treatment related changes in body weight or body weightgains noted for any animal during the study period. Any differences inbody weight or body weight gain were likely related to expectedbiological variation.

Individual concentrations of sialic acid versus time in Beagle dog serumfollowing IV or oral administration are shown in FIGS. 8A-8H.

TA-1

The background sialic acid levels were below zero for predose samples offive of the six animals, except for Animal No. 103, of which wasslightly above zero but below 20% of the LLOQ.

Following oral administration of TA-1 in prepared capsules at 3250 mg ofSA per animal, peak concentrations were observed ranging from 12.6 to40.8 μg/mL. T_(max) was observed at 2 hours postdose with the exceptionof Animal No. 103 (0.5 hours). The concentrations of sialic aciddecreased to levels below zero at 24 hours postdose for all six animals.The concentration of Animal No. 201 at 24 hour postdose (22.0989 μg/mL)was considered as aberrant and excluded from analysis, as it was a >LLOQvalue but following three <LLOQ samples which followed threequantifiable concentrations in the sampling sequence.

Towards the end of the sampling period, a decrease in sialic acidconcentrations was apparent, but the terminal elimination half-lifecould only be calculated for Animal Nos. 103, 201 and 203, ranging from1.39 to 1.49 hours.

The bioavailability of TA-1 was estimated to be ranging from 2.73% to6.76%, based on the individual AUC(0-t) value following IV and oraladministration.

All the predose urine samples had a measureable concentration of sialicacid while the data varied for each individual, ranging from 8.16 to25.1 μg/mL. The maximum excretion of sialic acid was observed forsamples collected 4-8 hours postdose, except for Animal No. 202 (0-4hours postdose). The total mass of sialic acid excreted in the urine wasequivalent to 0.43-3.56% of the doses of SA contained in TA-1.

The bioavailability of TA-1 was estimated to be ranging from 1.29% to39.1% based on the individual urinary percent excretion value followingIV and oral administration.

TA-2

The background sialic acid levels were below zero for predose samples ofAnimal Nos. 102 and 103 except for Animal No. 101, of which was slightlyabove zero but below 20% of the LLOQ.

Following oral administration of TA-2 in prepared tablets at 3250 mg ofSA per animal, Tmax was observed from 2.00 to 4.00 hours postdose withthe peak concentrations ranging from 7.98 to 13.7 μg/mL. Theconcentrations of sialic acid generally decreased after T_(max) tolevels below zero at 24 hours postdose, for all dosed animals. Theelimination half life of sialic acid was estimated to be 1.28 hour inAnimal No. 103. For Animal Nos. 101 and 102, the half-life could not beestimated as the measurable data were not enough to identify thetermination elimination phase.

The oral bioavailability of TA-2 was estimated to be ranging from 1.64%to 3.25%, based on the individual AUC(0-t) value following IV and oraladministration.

All the predose urine samples had a measureable concentration of sialicacid ranging from 13.5 to 34.8 μg/mL. The maximum excretion of sialicacid was observed for samples collected 4-8 hours postdose for allanimals. The total increase of sialic acid excreted in the urinepostdose was equivalent to 1.08-3.20% of the doses of SA contained inTA-2.

The bioavailability of TA-2 was estimated to be 2.53% and 3.73% forAnimal Nos. 102 and 103, respectively, based on the individual urinarypercent excretion value following IV and oral administration. Thebioavailability is 97.4% for Animal No. 101, which was markedly higherthan the other two animals due to its low percent excretion value of IVdoses.

TA-3

The background sialic acid levels were below zero for predose samples ofall three animals.

Following oral administration of TA-3 in prepared tablets at 3250 mg ofSA per animal, Tmax was observed from 2.00 to 4.00 hours postdose withthe peak concentrations ranging from 6.52 to 17.0 μg/mL. Theconcentrations of sialic acid generally decreased after Tmax to levelsbelow zero at 24 hours postdose for all three animals. However, thehalf-life could not be estimated for the three animals as the measurabledata were not enough to identify the termination elimination phase orthe extrapolation of the AUC to infinity represented more than 20% ofthe total area.

ÿhe oral bioavailability of TA-3 was estimated to be ranging from 1.46%to 4.14%, based on the individual AUC(0-t) value following IV and oraladministration.

Concentrations of sialic acid of all the predose urine samples werenoted to be slightly above LLOQ, ranging from 10.1 to 11.2 μg/mL. Themaximum excretion of sialic acid was observed for samples collected 4-8hours postdose for Animal Nos. 102 and 103, and 8-12 hours postdose forAnimal No. 101, respectively. The total increase of sialic acid excretedin the urine postdose was equivalent to 0.94-2.99% of the doses of SAcontained in TA-3.

The bioavailability of TA-3 was estimated to be 3.49% and 1.51% forAnimal Nos. 102 and 103, respectively, based on the individual urinarypercent excretion value following IV and oral administration. Thebioavailability is 85.0% for Animal No. 101, which was markedly higherthan the other two animals due to its low percent excretion value of IVdoses.

TA-4

In animals treated with TA-4, no sialic acid was measureable beyond 30minutes postdose.

Following oral administration of TA-4 in prepared tablets at 5000 mg ofSA per animal, most of the concentrations of sialic acid were below LLOQwith the exception of one dog (Animal No. 201), where the concentrationswere slightly above LLOQ at 4 and 6 hours postdose. Tmax was observedfrom 2.00 to 4.00 hours postdose with the peak concentrations rangingfrom 8.97 to 15.7 μg/mL. The concentrations of sialic acid generallydecreased after T_(max) to levels below zero at 24 hours postdose forall dosed animals. The half-life could not be estimated as themeasurable data were not enough to identify the termination eliminationphase.

The oral bioavailability of TA-4 was estimated to be ranging from 1.57%to 2.09%, based on the individual AUC(0-t) value following IV and oraladministration.

All the predose urine samples had a measureable concentration of sialicacid ranging from 6.4 to 42.6 μg/mL. The maximum excretion of sialicacid was observed for samples collected 8-12 hours postdose for AnimalNos. 201 and 203, and 0-4 hours for Animal No. 202, respectively. Thetotal increase of sialic acid excreted in the urine postdose wasequivalent to 0.54-1.93% of the doses of SA contained in TA-4.

The bioavailability of TA-4 was estimated to be 1.42% and 2.55% forAnimal Nos. 202 and 203 based on the individual urinary percentexcretion value following IV and oral administration. Thebioavailability of Animal No. 101 could not be estimated as the massexcreted of sialic acid postdose was set to be zero when corrected bypredose data.

TA-5

The background sialic acid levels of all animals were below zero forboth predose samples and 15 minutes postdose.

Following oral administration of TA-5 in prepared tablets at 5000 mg ofSA per animal, most of the concentrations of sialic acid were below LLOQwith the exception of Animal No. 201, of which were slightly above LLOQat 4 and 6 hours postdose. T_(max) was observed from 4.00 to 6.00 hourspostdose with the peak concentrations ranging from 7.79 to 15.3 μg/mL.The concentrations the sialic acid generally decreased to levels belowzero at 24 hours postdose for all three animals. However, the half-lifecould not be estimated for the three animals as the measurable data werenot enough to identify the termination elimination phase.

The oral bioavailability of TA-5 was estimated to be ranging from 1.47%to 1.96%, based on the individual AUC(0-t) value following IV and oraladministration.

Concentrations of sialic acid of all the predose urine samples rangedfrom 2.27 to 23.6 μg/mL. The maximum excretion of sialic acid wasobserved for samples collected 4-8 hours postdose for Animal Nos. 202and 203, and 0-4 hours postdose for Animal No. 201, respectively. Thetotal increase of sialic acid excreted in the urine postdose wasequivalent to 0.02-1.70% of the doses of SA contained in TA-5.

The oral bioavailability of TA-5 was estimated to be 0.52% and 2.24%,for Animal Nos. 202 and 203, respectively, based on individual urinarypercent excretion value following IV and oral administration. Thebioavailability of Animal No. 101 could not be estimated as the massexcreted of sialic acid postdose was set to be zero when corrected bypredose data.

TA-6

The serum levels of sialic acid were below zero for predose samples ofmost animals except for one dog, Animal No. 102, of which was slightlyabove zero but below 20% of the LLOQ.

Following an intravenous dose of 25 mg/kg of TA-6, the concentrations ofsialic acid decreased quickly to levels below LLOQ at the timepoint of 4hours postdose, and then to levels below zero at the timepoint of 8hours postdose, for all six animals. The concentrations generallydeclined in animals, except that Animal No. 203 exhibited much higherconcentrations at 6 hours postdose when compared to the previous timepoint. Sialic acid was eliminated in dogs with the half lives rangingfrom 0.56 to 1.40 hours.

The concentrations of sialic acid in the urine varied between eachanimal. It was noted that the sialic acid levels in urine collectedpostdose were lower than predose for Animal No. 201, with two of thethree concentrations of postdose samples detected as below zero.

The IV dose resulted in 72.4-87.7% of the administered dose beingexcreted in the urine of five of the six animals. One dog (Animal No.101) demonstrated excretion of only 1.1% of the applied dose.

The individual urinary percent excretion of TA-6 was used to adjust thedata of oral doses to account for the fraction of sialic acid absorbed(Fu, %). From the data reported herein, Animal No. 101 were observed tohave much lower urinary excretion postdose after IV dose, which resultedin a markedly higher value of bioavailability estimated for its oraldoses when compared with other animals in the same group.

In summary, after oral administration of TA-1 through TA-5, low sialicacid levels were detected in serum, most of which fell below the limitof quantitation. Peak concentrations ranging from 6.52 to 40.8 μg/mLwere observed from 0.5 to 6 hours postdose. Sialic acid was eliminatedwith a half-life of 0.56 to 1.40 hours, calculated based on the serumconcentration data from intravenous dose of TA-6. The bioavailabilitywas estimated based on the individual AUC(0-t) value following IV andoral administration. The pharmacokinetic parameters estimated for sialicacid in serum are presented in Table 21 to Table 23.

TABLE 21 Pharmacokinetic parameters estimated for sialic acid in serumRange Test Dose Level Animal Cmax Tmax AUC(0-t) Fs Article (mg/kg)Number (μg/mL) (h) (μg *h/mL) (%) TA-1 353-445 101-103 15.8-40.8 0.5-2  54.1-128  3.43-6.76 346-508 201-203 12.6-20.5 2 43.5-66.7 2.73-3.20 TA-2361-451 101-103 7.98-13.7 2-4 26.5-62.4 1.64-3.25 TA-3 353-439 101-1036.52-17.0 2-4 23.1-77.6 1.46-4.14 TA-4 538-746 201-203 8.97-15.7 2-438.3-74.4 1.57-2.09 TA-5 543-781 201-203 7.79-15.3 4-6 35.7-73.61.47-1.96 TA-6 25 101-103 — — 106-123 — 25 201-203 — — 108-120 — — = notapplicable.

TABLE 22 Pharmacokinetic Parameters of Sialic Acid in Beagle Dog SerumFollowing Oral Administration Dose Test Level Animal Cmax Tmax AUC(0-t)MRT(0-t) T½ AUC(0-inf) MRT(0-inf) Fs Article (mg/kg) Number (μg/mL) (h)(μg *h/mL) (h) (h) (μg *h/mL) (h) (%) TA-1 353 101 15.8 2.00 54.1 2.63 aa a 3.43 406 102 26.3 2.00 68.4 2.45 a a a 3.44 445 103 40.8 0.50 1282.07 1.41 137   2.45 6.76 TA-1 508 201 20.5 2.00 66.7 2.51 1.49 68.62.72 2.73 346 202 14.2 2.00 51.9 2.39 a a a 3.20 357 203 12.6 2.00 43.53.00 1.39 45.4 3.29 2.81 TA-2 361 101 7.98 4.00 26.5 3.51 a a a 1.64 392102 13.7 4.00 62.4 3.76 a a a 3.25 451 103 11.9 2.00 50.0 3.19 1.28 52.33.48 2.60 TA-3 353 101 6.52 4.00 23.1 3.65 a a a 1.46 382 102 17.0 4.0077.6 3.80 a a a 4.14 439 103 11.9 2.00 30.6 1.84 a a a 1.64 TA-4 746 20115.7 4.00 74.4 4.41 a a a 2.07 538 202 9.69 2.00 52.6 3.91 b b b 2.09562 203 8.97 2.00 38.3 3.37 a a a 1.57 TA-5 781 201 15.3 6.00 73.6 4.59a a a 1.96 543 202 8.20 6.00 46.4 4.70 a a a 1.83 562 203 7.79 4.00 35.74.58 a a a 1.47 a: parameter was not reportable due to the measurabledata were not enough to identify the termination elimination phase. b:parameter was not reportable due to the extrapolation of the AUC toinfinity represented more than 20% of the total area.

TABLE 23 Pharmacokinetic parameters of sialic acid in beagle dog serumfollowing IV administration Dose Test Level Animal AUC(0-t) MRT(0-t) T½AUC(0-inf) MRT(0-inf) CL Vd Article (mg/kg) Number (μg *h/mL) (h) (h)(μg *h/mL) (h) (mL/min/kg) (L/kg) TA-6 25 101 112 0.82 0.56 113 0.843.70 0.18 25 102 123 1.06 1.40 139 1.65 3.00 0.36 25 103 106 0.79 0.66108 0.84 3.87 0.22 TA-6 25 201 120 1.08 0.86 121 1.13 3.44 0.26 25 202117 1.27 0.99 119 1.35 3.51 0.30 25 203 108 1.18 0.58 113 1.43 3.67 0.18

All the predose urine samples had a measureable concentration of sialicacid and this was used to correct the total increase in urinaryexcretion of sialic acid at 12 hours postdose. In contrast to serum,most of the concentrations detected in urine samples exceeded the limitof quantitation. The urinary excretion of sialic acid, as a percent ofdose administered was estimated for each dosing occasion. Based on theassumption that the amount of drug excreted in urine after oraladministration was a reflection of the dose absorbed, thebioavailability of sialic acid was determined based on the percentexcretion value following IV and oral administration. The urinaryexcretion parameters estimated for sialic acid are presented in Table 24to Table 26

TABLE 24 Urinary excretion parameters estimated for sialic acid RangeTest Dose Level Animal Percent Excretion Fu Article (mg/kg) Number (0-12hr) (%) (%) TA-1 353-445 101-103 0.43-3.56 2.47-39.1 346-508 201-2030.93-2.57 1.29-2.17 TA-2 361-451 101-103 1.08-3.20 2.53-97.4 TA-3353-439 101-103 0.94-2.99 1.51-85.0 TA-4 538-746 201-203 0.54-1.931.42-2.55 TA-5 543-781 201-203 0.02-1.70 0.52-2.24 TA-6 25 101-1031.11-87.7 — 25 201-203   0-75.7 — — = not applicable.

TABLE 25 Urinary excretion of sialic acid in beagle dog following oraladministration Dose Dose Mass Excreted Percent Excretion Test (mg/ LevelAnimal Mass Excreted (μg) (0-12 hr) (μg) (0-12 hr) Fu Article animal)(mg/kg) Number Predose 0-4 hr 4-8 hr 8-12 hr Corrected (%) (%) TA-1 3250353 101 4652.55 149.630 13342.9 4314.71 17807.2 14085.2 0.43 39.1 406102 2448.73 549.029 65305.8 4816.71 70671.5 68712.5 2.11 2.47 445 1034206.50 313.356 101372 17403.2 119089 115724 3.56 4.06 TA-1 3250 508 2012372.90 0 85288.8 0 85288.8 83390.5 2.57 n/a 346 202 1177.24 21908.6319.025 8998.13 31225.8 30284.0 0.93 1.29 357 203 1966.81 17.302353674.5 1286.44 54978.2 53404.8 1.64 2.17 TA-2 3250 361 101 4083.30139.071 37257.4 942.062 38338.5 35071.9 1.08 97.4 392 102 4244.8514977.6 76498.4 15826.0 107302 103906 3.20 3.73 451 103 4006.52 1003.4673487.3 848.833 75339.6 72134.4 2.22 2.53 TA-3 3250 353 101 5310.076980.87 482.029 27399.5 34862.4 30614.3 0.94 85.0 382 102 4269.4933345.4 64010.5 3337.18 100693 97277.5 2.99 3.49 439 103 2936.63 1901.4042627.6 788.657 45317.7 42968.3 1.32 1.51 TA-4 5000 746 201 2767.70526.991 368.095 28316.5 29211.6 26997.4 0.54 n/a 538 202 5621.96 29695.1709.442 25589.5 55994.0 51496.5 1.03 1.42 562 203 4074.90 385.99173.4542 99426.6 99886.0 96626.1 1.93 2.55 TA-5 5000 781 201 1178.211269.32 372.873 63.4198 1705.61 763.046 0.02 n/a 543 202 966.212 303.20419168.7 281.709 19753.6 18980.7 0.38 0.52 562 203 2479.44 443.88163164.8 23357.0 86965.7 84982.2 1.70 2.24 n/a = not applicable

TABLE 26 Urinary excretion of sialic acid in beagle dog following IVadministration Dose Mass Excreted Percent Excretion Test (mg/ Dose LevelAnimal Mass Excreted (μg) (0-12 hr) (μg) (0-12 hr) Article animal)(mg/kg) Number Predose 0-4 hr 4-8 hr 8-12 hr Corrected (%) TA-6 227.5 25101 256.405 1941.56 509.021 274.902 2725.48 2520.36 1.11 202.5 25 1023508.71 161656 10622.6 4086.45 176365 173558 85.7 182.5 25 103 6124.14105.980 158924 5859.68 164890 159990 87.7 TA-6 162.5 25 201 3085.53267.937 0 0 267.937 0 0 237.5 25 202 1977.36 165111 400.537 8095.96173607 172026 72.4 227.5 25 203 1487.66 119734 50377.7 3394.26 173506172316 75.7

In conclusion, the pharmacokinetics of sialic acid in differentformulations following a single oral or intravenous dose in the beagledog were estimated, based on the serum and urine concentration data inthis study.

Example 11 Relative Therapeutic Effect of Continuous Vs. Once Daily OralExposure to Sialic Acid Replacement for 28 Days in HIBM Mice

The study is designed to assess the benefit of continuous exposure andsubstrate replacement with sialic acid versus episodic exposure tosialic acid. The use of sialic acid to produce glycoproteins andglycolipids is a continuous biologic process and stores of sialic acidintermediates are believed to be small. In addition, oral boluses ofsialic acid are likely to result in rapid urinary clearance. Bycomparing oral boluses with continuous exposure we can assess therelative benefit of providing continuous delivery of sialic acid, thatmight be achievable in humans with an extended release ER formulation.ALZET pumps will allow us to provide a continuous level of drug exposureto compare with oral bolus treatment. The effect of the therapy onbiochemical normalization of sialylation and effects on muscle pathologyand function are assessed to determine the impact of continuous versusbolus therapy.

Experimental Design

The experimental design will compare one group of mice treated with onceper day bolus of sialic acid at 200 mg/kg, with two dose levels ofsialic acid provided by continuous IP administration using Alzetminipumps. Female HIBM mice are used for this study with five mice perstudy group. The dose levels chosen for the minipump animals are 5 mg/kgwhich would be about equivalent to 200 mg/kg orally with a 2.5% rate ofabsorption, and 20 mg/kg, which would be about 10% rate of absorption.Due the limited number of animals, there are no control animals. Aprevious experiment in similarly aged mice would provide potentialcomparative control animals for this experiment. The design will capturebaselines for easily accessible samples, including free sialic acid,creatine kinase in blood, polysialylated NCAM (PSA-NCAM) from blood, andtotal sialylated proteins in blood if possible. The animals are treatedfor 1 month, and sampled as intervals as shown. At the end of the month,the animals are taken down with blood for the same assays above, as wellmuscle tissues for NCAM westerns, frozen sections for surfacessialylation, and samples for biochemical analysis of bound and freesialic acid. By comparing the degree of sialylation in the blood and inthe tissues, it is determined if bolus or continuous exposure aresuperior. The total dose is checked by measuring free sialic acid in theblood and by measuring sialic acid in the urine.

Test Article 1: Bolus: Group 1

Oral formulation of sialic acid 4 mg/ml is administered at a dose of 200mg/kg/day. The volume of injection is adjusted by individual body weightand dose concentration. Approximate dose volume per day is 250 uL.

Test Article 2: Pumps: Group 2

IP formulation of sialic acid 37.5 mg/ml is administered at a dose of 5mg/kg/day. The volume of injection is 100 uL total per pump.

Test Article 3: Pumps: Group 3

IP formulation of sialic acid 150 mg/ml is administered at a dose of 20mg/kg/day. The volume of injection is 100 uL total per pump.

Animal Care and Housing

Post Alzet pump placement each mouse is housed individually for accurateclinical observations and safety of the animal. Oral bolus group may behoused together in one cage. Each cage will receive water ad libitum.Animals are examined during the working day for morbidity and mortality.Body weights are recorded prior to the first dose administration andonce weekly thereafter. Animals are fasted prior to pump placement andprior to blood collection and necropsy days. Animals are fed 4 hrs postdose but fasted for no longer than 24 hours.

Alzet Pumps: Preparation and Implantation

Alzet pump model 1004 with a flow rate of 0.11 uL/hr for 28 days is usedand the manufacturer's recommendations regarding handling, priming andfilling the Alzet pumps (WorldWideWeb:alzet.com/products/guide_to_use/filling.html) is followed

Animals are anesthetized using isoflurane inhalation prior to Alzet pumpplacement. For intraperitoneal implantation, the following steps areperformed:

1. Once the animal is anesthetized, the skin over the implantation siteis shaved and washed.2. A midline skin incision, 1 cm long, in the lower abdomen under therib cage is made.3. The musculoperitoneal layer is carefully tented up to avoid damage tothe bowel. The peritoneal wall directly beneath the cutaneous incisionis incised.4. A filled pump, delivery portal first, is inserted into the peritonealcavity.5. The musculoperitoneal layer is closed with 4.0 absorbable suture inan interrupted or continuous pattern, taking care to avoid perforationof the underlying bowel.6. The skin incision is closed with 2 or 3 wound clips or interruptedsutures.

Blood Sample Collection and Processing

Dosing and sampling are performed as follows:

Group 1: Oral Bolus: N=5 miceFast prior to PK sampling

Days 1, 14, 21, 28: Record BWs

PK Sampling: As per table belowDays 1, 14, 21, 28: collect predose CK, total sialylation and NCAMsamples

Days 1-28: Single Oral Dose

Day 29: final blood collection (CK, total sialylation and NCAM samples)and necropsy; assay a set of muscles for surface sialylationGroups 2 and 3: IP Alzet Pump: N=5 mice for each groupFast prior to PK samplingDay −1: implant pumps

Days 1, 14, 21, 28: Record BWs

PK Sampling: As per table below.Days 1, 14, 21 collect predose CK, total sialylation and NCAM samplesDay 29: final blood collection (CK, total sialylation and NCAM samples)and necropsy.

PK Sample Collection Schedule GROUP 1: Oral Bolus Group SampleCollection Time Points (Time Post Dose) on Day 1, 14 and 28 Group No.Pre 1 hr 1 x x GROUPS 2 AND 3: Pump Group Sample Collection Time Points(Time Post Dose) on Days 1, 14, 28 Group No. Pre 2 x

Blood samples are collected at the prescribed time points/days asdescribed above.

PK samples: Approx. 25 uL whole blood/time point. Blood samples arecollected in serum tubes or in a 96 well plate. The blood is allowed toclot in the tubes at RT for 1 hr prior to centrifugation. Serum samplesare stored at −80° C. for the duration of the study.

CK samples: At least 200 uL whole blood/time point is collected for CKclinical chemistry.

NCAM samples: Approx. 125 uL whole blood/time point. Blood samples arecollected in serum separator tubes. The blood is allowed to clot in thetubes at RT for 1 hr prior to centrifugation. Serum samples are storedat −80° C. for the duration of the study.

Tissue Collection and Processing

After the last blood sample collection, mice are euthanized and tissuesamples are collected. Muscle is analyzed for sialylation via lectinstaining and Western Blot. Tissues are analyzed with H& EStaining/trichrome and Congo Red.

The following tissues are collected on Day 29:

Quadracep muscleHamstring muscleBicep muscleTricep muscleTibialis anterior

For the tissue samples, three pieces for each muscle are needed:

1. 1 piece fixed in 10% Neutral Buffered Formalin, for paraffinembedding & sectioning and staining with H&E, Congo Red and Masson'sTrichrome.2. 1 piece frozen in a microfuge tube at −80 degrees for frozen sectionsfor lectin staining.3. 1 piece weighed and frozen at −80 degrees in a microfuge tube forextract preparation for N-CAM and other westerns as well as biochemistryof sialylation (total bound sialic, GM3 levels.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is apparent to those skilled in the art that certainminor changes and modifications will be practiced. Therefore, thedescription and examples should not be construed as limiting the scopeof the invention.

1.-39. (canceled)
 40. An extended release pharmaceutical formulation fora human patient in solid oral unit dosage form comprising: a)N-acetylneuraminic acid (NeuAc), or a pharmaceutically acceptable salt,or solvate thereof; b) one or more hydrophilic polymers; c) one or moreanionic, pH-dependent, gel-forming polymers; and d) one or morehydrocolloid polymers or one or more cationic polymers; wherein theformulation provides a therapeutically effective and a steady plasmaconcentration of NeuAc over a period of greater than 6 hours.
 41. Theextended release pharmaceutical formulation of claim 40, comprisingabout 20% to about 80% w/w NeuA.
 42. The extended release pharmaceuticalformulation of claim 40, comprising about 30% to about 60% w/w NeuA. 43.The extended release pharmaceutical formulation of claim 40, comprisingabout 40% to about 45% w/w NeuA.
 44. The extended release pharmaceuticalformulation of claim 40, wherein the one or more hydrophilic polymerscomprise one or more water-swellable, pH independent polymers selectedfrom the group consisting of hypromellose; hydroxypropyl ethylcellulose; hydroxypropyl cellulose; hydroxyethyl cellulose; and methylcellulose.
 45. The extended release pharmaceutical formulation of claim41, wherein the one or more water-swellable, pH independent polymerscomprise hypromellose.
 46. The extended release pharmaceuticalformulation of claim 40, comprising about 20% to about 50% w/w of one ormore hydrophilic polymers.
 47. The extended release pharmaceuticalformulation of claim 40, comprising about 20% to about 30% w/w of one ormore hydrophilic polymers.
 48. The extended release pharmaceuticalformulation of claim 40, wherein one or more anionic, pH-dependent,gel-forming polymers comprise an alginate salt.
 49. The extended releasepharmaceutical formulation of claim 40, wherein the alginate saltcomprises sodium alginate.
 50. The extended release pharmaceuticalformulation of claim 40, comprising about 15% to about 30% w/w of one ormore anionic, pH-dependent, gel-forming polymers.
 51. The extendedrelease pharmaceutical formulation of claim 40, comprising about 20% toabout 25% w/w of one or more anionic, pH-dependent, gel-formingpolymers.
 52. The extended release pharmaceutical formulation of claim40, wherein the one or more hydrocolloid polymers comprise one or morecarrageenans.
 53. The extended release pharmaceutical formulation ofclaim 47, wherein the one or more carrageenans comprise lamdacarrageenan.
 54. The extended release pharmaceutical formulation ofclaim 40, comprising about bout 1% to about 10% w/w of one or morehydrocolloid polymers or one or more cationic polymers.
 55. The extendedrelease pharmaceutical formulation of claim 40, comprising about bout 1%to about 5% w/w of one or more hydrocolloid polymers or one or morecationic polymers.
 56. The extended release pharmaceutical formulationof claim 40, further comprising about 1% to about 10% of a mixture ofmicrocrystalline cellulose and colloidal silicon dioxide.
 57. Theextended release pharmaceutical formulation of claim 40, furthercomprising about 0.1% to about 1% of one or more lubricants.
 58. Theextended release pharmaceutical formulation of claim 57, wherein the oneor more lubricants comprise magnesium stearate.
 59. The extended releasepharmaceutical formulation of claim 40, which provides a therapeuticallyeffective amount of sialic acid over a period of about 8 to about 10hours.
 60. The extended release pharmaceutical formulation of claim 40,which is a tablet or capsule.
 61. The extended release pharmaceuticalformulation of claim 40, wherein the formulation is administered two,three or four times a day.